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Compass Software for Peggy User Guide

1. 326 Stacking Multiple Electropherograms 327 Overlaying Multiple Electropherograms 328 ZOO aTa A EE aes nadie 329 Customizing the Data Display 04 330 Selecting Data Viewing QDIONS erea 339 Adding and Removing Baseline Points 345 Selecting the X Axis pl RANGE 0600 0 eee 346 COSC INONT CS secre nenahinig A E 347 Compass Analysis Settings Overview 4 348 Aavanced ANGIVSIS SENGS yiri reenn 350 SIGNOGIGS SCHINGS ss serii trr nE EEr 35 SOIMDICS EIN S E E E E EEE 351 ROEE a aE EEE E TAE 39 Compass Software for Peggy User Guide Advanced Analysis Settings Groups 552 Creating a New Analysis Group 0665 302 Changing the Default Analysis Group 353 Modifying an Analysis Group 00 6 0 cece 354 Deleting an Analysis Group 2 0 6c c cece eee 354 Applying Analysis Groups to Specific Run Data 354 Imag ANIGIYSIS SCTLINGS tise warns a ga ake Raven S57 EXPOSTO SENIN 40 2 40575 waswuniaei nade t Wo acute 550 Changing the Sample Data Exposure BIC Cee eee ee ey eee T E renee 359 Peak Fit Analysis Settings 0 0 0c cece cece cece 360 KONGE CUNO S aus en ohach E E N 361 BOS CHC SENGS wes sac i err Err riir EE 301 PCOK FING ITONG S Arr 361 Peak Fit Analysis Settings Groups 361 Creating NCW Peak Fil GIOUD rrrr eiin eins 362 Changing the Default Peak FitGroup 363 Modifying aPeak Fit GrOUD 0 0 c cece eee 363 ERUNGO
2. dll Compass Software for Peggy User Guide page 4 Chapter 1 Getting Started with Compass Run Summary Screen The Run Summary screen is used to monitor status of a run in progress watch movies of the separation in the capillaries and view current and voltage plots for each run Pa 2012 03 05_11 51 19_HelaControlERKassay Compass nA File Edit Instrument Window Help Assay 5 Run Summary i Analysis Separation gt lt IV Plot m Run 2012 03 05_11 51 19_HelaControlERKassay TY Status m Run 2012 03 05_11 51 19_HelaControlERKassay Path C Users pfung Documents ProteinSimple 2012 03 05 11 51 19 HelaControlERKassay cl Assay HelaControlERKassay Schedule Overlapping with hold Instrument PLOO04 PLO004 Started Mon 11 56 AM Mar 5 2012 PST Completed Tue 6 35 AM Mar 6 2012 PST Cycle Sample Sep Hold 1 k a a Detect Results T rO 20 30 D po 1 W we amp D 7 x 11 56AM 12 01PM 1 10PM 6 10PM 6 36PM 8 45PM 9 53PM 10 27 PM VY Very DEVS 3 00 Gl amt 2 Lii ai a 1 10PM 1 14PM 2 24PM 7 27PM 7 53PM 10 02PM 11 10PM 11 44 PM TY Very 2 F 3T Gal r r z Lii 1 i l 2 24PM 2 28PM 3 37PM 8 46PM 913PM 11 22PM 12 30AM 1 04AM I TY ror r 3 T m Fae 4 Lii ee tnt al pn oh fos ames 3 37PM 3 42PM 4 51PM 10 04PM 10 30PM 12 39AM 1 47AM 2 21AM TY Tan r yan m po 5 ii t as i D gt lt 0 Id B 4 51PM 4 56PM 6 05PM 10 58PM 11 24PM 1 33AM 2 42AM
3. While ProteinSimple recommends using the default assay detection profile users can change the profile exposure times and exposure sequences if needed To do this 1 Select the exposures cell in the Protocol pane and click the button or double click in the cell The fol lowing screen will display eS Detection Profile R 3 m S ig E Exposure sec 30 0 60 0 120 0 240 0 480 0 Compass Software for Peggy User Guide Copying Protocols and Templates page 89 Each row represents an individual exposure that will be taken during the run a To change an existing exposure time Click in the exposure cell and enter a new time in seconds 4 Detection Profile Exposure sec 30 0 60 0 120 0 240 0 Fr b To delete an existing exposure Select a type or exposure cell and click Remove c To add anew exposure Select Add A new exposure will be added to the end of the list Click in the exposure cell and enter an exposure time in seconds 2 Click OK to save and exit Copying Protocols and Templates The steps and parameters in the Protocol pane can be copied and d into other documents as can the graphic image of the annotations in the Template pane Copying an Assay Protocol 1 Click on the Protocol tab 2 Select Edit in the main menu and click Copy 3 Open a document Word Excel etc Right click in the document and select All assay protocol steps and parameters will be copied into the document as
4. Open Run j Add Run b Close Close All Save 5500 4 Save As 5000 4 Export Tables t poe Export Spectra p Text Format Exit Bon To export data in txt format Select Text Format Plots will be exported in one file for all capil laries To export data in cdf format Select Andi Format Plots will be exported in one file per capil lary 2 Select a directory to save the files to and click OK Data will be exported in the selected format Changing Sample Protein Identification Compass allows you to customize what sample proteins are reported in the results tables by making manual adjustments in the electropherogram or peaks table Adding or Removing Sample Data 1 Click Show Samples in the View bar 2 Click Single View in the View bar 3 Click on the row in the experiment pane that contains the sample you wish to correct then click the Graph tab To remove a peak from the data Right click the peak in the electropherogram or peaks table and select Remove peak Compass will no longer identify it as a sample peak in the electro pherogram and the peak data will be removed in the results tables Compass Software for Peggy User Guide Changing Sample Protein Identification page 313 Samples 1400 1300 1200 1100 1000 perki Erk pERK2 ppERK2 Chemilumines cence Zoom Out 5 0 51 52 a3 DA 55 56 57 58 59 60 61 Eo 63 6 4 E5 66 67 6 8 Remove Pea
5. To view standards data Click Show Standards in the View bar or select View in the main menu and click Standards File Edit View Instrument Wi ndow Help Assay C3 Run Summary oes 2 Primary Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki w o c w o w e E u 440 420 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 x ANE Graph BA Image E Lane Standards tel O 7o Position Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki FP FF Fe wWWwWssnsnwnwnwnsnwnwnwnwnnnn nnn Ni NN NIN RP RF RP RP RP RP RP RP RP eR Re Cap Peak Position C2 1 C2 1 C2 1 1 4 9 275 382 870 Height 393 3 27 2 18 8 Compass Software for Peggy User Guide Viewing Run Data page 149 Data in this view is for the fluorescent standards only These standards are premixed with each sample prior to analysis Graph view data displays electropherograms in fluorescence y axis and position x axis Lane view data displays fluorescent standards only Image view data displays
6. Ty T T Gl bow 10 2 30 A eS 11 56AM 12 01PM 1 10PM 6 10PM 6 36PM 8 45PM 9 53PM 10 27PM Ty T cts Lal Prom ae 1 10PM 1 44PM 2 24PM 7 27PM 7 53PM 10 02PM 11 10PM 11 44PM WwW 1 2 TY War r 3 T Bal Eo 3 2a O ae a a G 2 24PM 2 28PM 3 37PM 846PM 9 13PM 11 22PM 12 230AM 1 04AM TY rer PT 3 r r 4 e a a 3 37PM 3 42PM 4 51PM 10 04PM 10 30PM 12 39AM 1 47AM 2 21AM TY rer 2 60 yar m l r C 5 WM te LS gt iid B 4 51PM 4 56PM 6 05PM 10 58PM 11 24PM 1 33AM 2 42AM 3 15AM TY rer 2 60 3 07 Gl EZERS 5 iL a Uri 6 05PM 6 09PM 7 18PM 11 39PM 12 05AM 2 14AM 3 22AM 3 56AM TY Tan r 3 T Gl Fess 7 Lii i s s i Verri 719PM_ 7 23PM 832PM 1220AM 1246AM 255AM 4 03AM 4 37AM TY nTa T 3T Cl nes g LL eee 8 32PM 8 37PM 9 46PM 1 09AM 1 36AM 3 44AM 4 53AM 5 26AM 3 For each capillary verify that the standards visibly separated Each fluorescent point in the capillary rep resents one of the three sizing standards Step 2 Checking Fluorescent Sizing Standards To verify the standards are identified correctly 1 Click the Analysis screen tab 2 Click Show Standards in the View bar Verification that the standards have been correctly identified can be done in either the graph or lane panes Compass Software for Peggy User Guide Checking Your Results page 165 Graph Pane a Click Single View in the View bar b C
7. 2 If needed well assignments can be modified as described below Any row assignments changed in the Layout pane are updated in the Protocol pane automatically To move a reagent row to another location Click the row in the Layout pane then drag and drop it on the new location The row from which it was moved will be reassigned as empty Layout F i A B a C G D 4 D E E F F G G H H I J 4 J E K L L M i M N Separation Matrix H o l Slacking Mabrix o P P Compass Software for Peggy User Guide page 28 Chapter 2 Size Assays Toinsert a new row Click the row below where the new one should be inserted then click Insert an empty row circle icon in the Layout pane toolbar A new row will be inserted above the selected row Eau XOS 12 3 4 ie Insert an empty row 4 HEE j k ie a e a a e E e e A a To insert a sample row Click an empty row or the row below where the new sample row should be inserted then click Insert a sample row S icon in the Layout pane toolbar A new sample row will be added in the empty row or inserted above the selected row E i Layout a Oo A nm 0AP Zt TONMoOoab A7 Toinsert a luminol row Click an empty row or the row below where the new luminol row should be inserted then click Insert a luminol row L icon in the Layout pane toolbar A new luminol row will be added in the empty row or inserted above the selected row
8. High High High High High High High High High High High High High High Low p Low p Low p Low p Low p Low p Low p Low p Low p Low p Low p Low p Low p Low p Low p Low p 1 f Experiment E Primary ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 RFPrRPrPrRPrP RFP RP RP RP RP RP RP RP RP RP RP RP RP RP RP RP RP RP PP BP PP PP eB eR 4 die Image Te a o Samples og o i w o no w E E 3 E w O 200 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 ppErk1 pErk1 Erk1 pERK2 ppERK2 50 St S2 amp 3 54 55 55 S 5A 55 60 61 G2 6 amp 3 64 655 G6 G7 BB 83 FO 1 s 1 Capillaries High pho ERK1 2 Cid 3 ppERK2 607 5 805 711 2 5864 i Cap Peak Name Position Area Area Width S N 62 4 0 0239 1538 7 Data in this view is for immunodetected sample proteins only Graph view data displays electropherograms in chemiluminescence units y axis and pl x axis Lane view data displays immunodetected sample proteins only Image view data displays immunodetected sample proteins only Re
9. To move a reagent row to another location Click the row in the Layout pane then drag and drop it on the new location The row from which it was moved will be reassigned as empty Fimo x OSB 12 3 4 i gt TOTErAt TAmoage DOFErAC TEAMNMSAGE f Toinsert a new row Click the row below where the new one should be inserted then click Insert an empty row circle icon in the Layout pane toolbar A new row will be inserted above the selected row Compass Software for Peggy User Guide page 70 Chapter 4 Charge Assays Eom xi OJB 12345 1 75 Insert an empty row R 20 nm 0T P ma eA e r To insert a sample row Click an empty row or the row below where the new sample row should be inserted then click Insert a sample row S icon in the Layout pane toolbar A new sample row will be added in the empty row or inserted above the selected row E Layout a x OE1 23485 L Ae 0 nma 0 Re EOMMSAae Toinsert a luminol row Click an empty row or the row below where the new luminol row should be inserted then click Insert a luminol row L icon in the Layout pane toolbar A new luminol row will be added in the empty row or inserted above the selected row Layout B xO 123 4 Primary Ab Tir Secondary Ab Tertiary Ab Tin Detection Zz G ITAAMoOoOeP h E Ze EOoOMmmooOoSb e Toinserta fourth or fifth incubation reagent Click an empty row or the row below where the new incu
10. 216 249 271 298 329 Compass Software for Peggy User Guide page 150 Chapter 8 Size Assay Data Analysis Data in this view shows capillary registration only Graph view data displays electropherograms in fluorescence y axis and position x axis Lane view data displays capillary registration only Image view data displays capillary registration only Registrations are highlighted in both the peaks and capillaries tables and marked with an R Because capillaries must be moved multiple times during the run their positions are tracked to account for potential changes in imaging position as they are moved to and from the separation block To do this one of the standards is used as a capillary registration An image of the standard used for registra tion is taken at the end of the separation step prior to chemiluminescent imaging The position of this peak in the registration image is then compared to the position of the same standard peak in the stan dards image The shift value listed in the peaks table is the change in pixel position between the two images Compass data is then aligned to account for any changes in capillary position For information on checking and identifying registration peaks see Step 3 Checking Capillary Regis trations on page 168 Compass Software for Peggy User Guide Viewing Run Data page 151 Selecting and Displaying Capillary Data You can choose to view data from one multiple o
11. ERK1 2 C1 1 3 297 R High ERK1 2 Cia 4 312 High ERK1 2 Cll 331 High ERK1 2 Cii 361 High ERK1 2 C11 872 High ERK1 2 Ci 898 R High ERK1 2 Cid 917 Hinh FRK1 1 1 945 5 Ifthe registration peak is not identified correctly right click the correct registration peak in the electro pherogram or peaks table and select Registration Force Compass will assign the new peak as the cap illary registration A lock icon indicating the registration was set manually will display next to the peak in the peaks table Compass Software for Peggy User Guide Checking Your Results page 301 NOTE To remove registration peak assignments that were made manually right click on the peak in the electropherogram or peaks table and click Registration Clear 6 Repeat the previous steps for the remaining rows in the experiment pane to make sure all registrations are identified correctly Step 4 Checking Samples All immunodetected sample proteins in the graph and lane panes will be labeled automatically with the cal culated protein pl NOTE The reported pl for immunodetected sample proteins in Compass may vary slightly trom predicted pls based on sample buffer and assay conditions To verify that sample proteins are identified correctly 1 Click the Analysis screen tab 2 Click Show Samples in the View bar Verification that sample proteins have been correctly identified can be done in
12. How Run Data is Displayed in the Analysis Screen 139 Experiment Pane Assay and Capillary PORTO NON irren a EEE A 139 Graph Pane Electropherogram Data 140 Image Pane Capillary Separation Image Data 141 Lane Pane Virtual Blot Like Image Data 142 Peaks Pane Calculated Results 143 Capillaries Pane User Specified Peak Names 144 KECA 12e e EEEIEE E AE 146 Switching Between Sample Standards and kegi trauomDalta VICWS reae nehes Aaa 146 Selecting and Displaying Capillary Data 15 Switching Between Single and Multiple Views of CC COON GCS oo a ssn ee shila a Bis E 155 Mang COD MANOA aeae apouee saat 59 Setting Run Data Display Filters 4 160 Compass Run Data Notifications and Warnings 162 CHECKING TOORE ernan AEE 163 Step 1 Review the Fluorescent Sizing Standards NOVAS EEE Sat E atom acres hale neta 163 Step 2 Checking Fluorescent Sizing STNG ONS PEE then ima Caen aa EE 164 Step 3 Checking Capillary Registrations 168 page iv Step 4 Checking the Ladder 0664 Step 5 Checking Samples 00 66 Step 6 Assigning Peak Names Optional GOCO I U raat ae ceca acne ae et EEES USINGIGIOUDS E E AE E E E VICWING Ta ICE aR EEEN Hiding or Removing Capillaries in Group PUN EENE E A aad Copying Data Views and Results Tables COVO DOUI VION Sisk 5 tots sipnsctite west nie Ruby Monee COPVING ICSUNES TOMES wee ninares roi ee Saving th
13. Primary Capillary Biotinylat Blocking 1 1 K562 anti E Cl 2 K562 anti E c13 K562 anti E C14 K562 anti E C15 anti E C1 6 rernm m NOTES Peaks that Compass names automatically with user defined peak name settings are color coded Information displayed for fluorescent standards and capillary registration data will be for identified stan dards or registration peaks Compass Software for Peggy User Guide How Run Data is Displayed in the Analysis Screen page 145 To view all rows Click the Capillaries tab then use the scroll bar or click Maximize in the upper right corner To resize columns Click the Capillaries tab Roll the mouse over a column border until the sizing arrow appears then click and drag to resize Capillaries table column descriptions are as follows Sample Sample name If sample names were entered in the assay template Assay screen those names will display here Otherwise Sample default name will display Primary Primary antibody name If primary antibody names were entered in the assay template Assay screen those names will display here Otherwise Primary default name will display Capillary Cycle and capillary number For example C1 3 indicates cycle 1 capillary 3 NOTE Peggy runs12 capillaries at a time in a cycle and is able to run up to eight cycles in an experiment The Information on cycle and capillary number is displayed in the Experiment tab P
14. T wa r zT Cl mnt 3 37 PM 3 42 PM 4 51PM 10 04PM 10 30PM 12 39AM 1 47AM 2 21AM D 1 20 7 f g 5 LI t z sa i 40 ip B 4 51 PM 4 56 PM 6 05PM 10 53PM 11 24PM 1 33AM 2 42AM 3 15AM TY 1 TY P pyan m r r 6 WW amp J St bmg 6 05 PM 6 09 PM 7 18PM 11 39PM 12 05AM 2 14AM 3 22 AM 3 56 AM TY Pry yaa 3 T m f uij t gt a 7 19 PM 7 23 PM 8 32PM 12 20AM 12 46AM 2 55AM 4 03 AM 4 37 AM TY ry rM zT m GER 8 32 PM 8 37 PM 9 46 PM 1 09 AM 1 36 AM 3 44 AM 4 53 AM 5 26 AM X 3 For each capillary verify that the standards visibly separated Each fluorescent point in the capillary rep resents one of the three sizing standards Step 2 Checking Fluorescent Sizing Standards To verify the standards are identified correctly 1 Click the Analysis screen tab 2 Click Show Standards in the View bar Verification that the standards have been correctly identified can be done in either the graph or lane panes Compass Software for Peggy User Guide Checking Your Results page 297 Graph Pane a Click Single View in the View bar b Click on the first row in the experiment pane then click the Graph tab Check that the electrophero gram has the appropriate number of fluorescent pl standard peaks for the pl Standard Ladder you are using They will also be identified with a green S in the peaks table The pl standards at the low and high end of the pl range in the electropherogram are at higher
15. jm A Sample Primary Cycle biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki Blocki Blocki in Blocki Blocki Blocki Blocki Blocki Blocki biotin ladder C3 1 Ldr 116 y 2 888888 40 MW kDa HE Peaks FHE Capillaries Sample Primary Cap Peak Name Position MW kDa Height Area Area Width S N biotin Blocki C3 1 1 Ldr12 350 1928 3 41626 20 3 biotin Blocki C3 1 Ldr 40 588 1514 9 33744 20 9 biotin Blocki C3 1 Ldr 66 701 1416 5 30665 20 3 biotin Blocki C3 1 Ldr 90 741 3438 1 65253 178 biotin Blocki C3 1 Ldr 116 777 2051 3 38733 17 7 biotin Blocki C3 1 Ldr 180 859 1064 0 26008 23 0 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 A If ladder peaks are not identified correctly they can be manually corrected as follows Ifan incorrect peak is identif
16. 40 0 t 37 5 90 10004 2 135 02 3 2 900 80 5 g 3255 800 4 t 30 0 70 170 o 127 5 600 4 t225 150 50 500 20 0 4 140 A 117 5 130 oa 115 0 120 30 300 112 5 1104 200 10 0 20 100 al 75 90 10 5 0 04 80 lt 25 0 500 1000 1500 2000 2500 o 500 100 41500 20o 2500 Time Seconds Time Seconds The blue Y axis and plot shows the run voltage in volts V and the red Y axis and plot shows the run current in micro amps uA The X axis displays time in seconds To zoom in on an area of the plot Hold the mouse button down and draw a box around the area with the mouse To zoom out Click Zoom Out in the upper right corner of the pane NOTE The IV plot for a run in progress will not be available until the separation step starts executing The plot is then displayed in real time Compass Software for Peggy User Guide page 118 Chapter 6 Run Status Switching Between Open Run Files If more than one run file is open you can switch between viewing the run information in each To do this 1 Click the down arrow in the run box Run Simple Western ERK Demo ka 2011 07 13_16 55 20_7 12 2011 Simple Western ERK Demo H0 Run Simple Western ERK Demo Path CAUsers OwnernDocuments Compass Runs simple Western ERR Demo chz Assay simple Western Demo Assay 2 Select the run you want to view from the drop down list Closing Run Files If more than one run file is open you can close just one file or all th
17. 49 biotin Blocki 59 biotin Blocki 29 S biotin Blocki 18 8 e FF Fe FFF ewww sswnwnsnnnsnnnnnnnnnn nnn FR rr RR RR RR eR If standards are not identified correctly they can be manually corrected as follows e Ifan incorrect peak is identified as a standard Right click the peak in the electropherogram or peaks table and select Not a Standard Compass should correctly reassign the remaining peaks as standards and update the peaks table Compass Software for Peggy User Guide page 166 Chapter 8 Size Assay Data Analysis To set an unidentified peak as a standard Right click the peak in the electropherogram or peaks table and select Force Standard Compass will assign the peak as a standard and cor rectly reassign the remaining standard peaks A lock icon indicating the standard was set man ually will display next to the peak in the peaks table NOTE To remove standards peak assignments that were made manually right click on the peak in the electropherogram or peaks table and click Clear c Repeat the previous steps for the remaining rows in the experiment pane to make sure all standards are identified correctly Lane Pane a Click Multiple View in the View bar b Click on the first row in the experiment pane then click the Lane tab Standards will be bands and identified with a green outline Check that there are three standard bands labeled Std 1 0 Std 12 0 and Std 180 0 They wil
18. Blocki Blocki Blocki Blocki Chemiuminescence BUNARA Chemibminescence BHR ABR Chemibminescence PELL Chemibminescence ELL w w w wwnwwnnnn nn rn NNN N NR eR w Primary Name Position MW kDa Height in Blocki Ldr 116 801 116 2032 5 Blocki Ldr 180 1046 4 gt e e FFF FW Ww Ww Ww WwW Data for the row s selected in the experiment pane will display as follows Electropherograms corresponding to the selected row s are highlighted and displayed in the graph pane Results for selected capillaries display in the peaks and capillaries tables and are highlighted The selected row s are highlighted in the image pane Compass Software for Peggy User Guide Viewing Run Data page 159 IE Graph 2 Image Lane Cycle I Samples Exp 300 All selected lanes display in the lane pane and lanes corresponding to the selected row s are highlighted E Graph 5 Image Lane PHB A 8 SEP EEE EE EES z 2 T ei ch a Q RS YP PF PN PP od oo Hiding Capillary Data Capillary data can be hidden from view if needed To do this 1 Click the Experiment tab Compass Software for Peggy User Guide page 160 Chapter 8 Size Assay Data Analysis 2 Select the rows you want to hide then right click on one of the selected rows and click Hide E Experiment E gt Sample Pri
19. EREL 2 Primary 2 Low phosphe HeLa 24 ERKI 2 Primary 2 Low phosphe HeLa 24 EREL 2 Primary 2 Low phospho HeLa 24 ERKI 2 Primary 2 Low phospho HeLa 24 EREL 2 Primary 2 Clicking the arrow next to a group lists the individual capillaries in the group and reported data for each cap illary Compass Software for Peggy User Guide Group Statistics Sample b Hi phospho HeLa 24 4 Hi phospho HeLa 24 rT F F F F F F F Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phosphe HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phospho HeLa Hi phosphe HeLa 24 Hi phosphe HeLa 24 Hi phosphe HeLa 24 Hi phosphe HeLa 24 Hi phosphe HeLa 24 Hi phosphe HeLa 24 Hi phosphe HeLa 24 Hi phosphe HeLa 24 if i i Bt rat s H Capillary Groups lu Group Plot Primary ERKL 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERE1 2 Primary 1 ERK1 2 Primary 1 ERKL 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERE1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERK1
20. ERK1 2 Low p ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Hi ph ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Lm E m Chemiluminescence Beebe eae 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 7 0 HE Capillaries Primary Cap Peak Name Position Area Area Width Wwoweeoewnwogsnwnwnennn nn nr NNN NN FP RP RP RP RP RY eR eR Compass Software for Peggy User Guide page 378 Chapter 9 Charge Assay Data Analysis Standards Settings The standards analysis settings page lets you view and change the pl and position for fluorescent standards and set the registration peaks To access these settings select Edit in the main menu and click Analysis then click Standards in the options list NOTE Settings can be modified in an assay prior to starting a run or ina run file once the run has finished executing Analysis changes made to an executing run will not be saved to the final run file Default Analysis type filter text Standards Advanced Images Analysis Settings Peak Fit Peak Names Standards Std Ladder1 premix pI 3 10 Std Ladder1 premix pl 3 10 Fluorescent Peaks Std Ladder premix pl 4 7 Std Ladder premix pl 5 8 pl Std Ladder4 premix pl 5 6 4 200 Standard 49 350 6 520 64 550 73 650
21. High phos ERKI 2 1 Low phosp ERKI 2 1 High phos ERKI 2 1 Low phosp ERK1 2 1 High phos ERKI 2 1 Low phosp ERKI 2 1 High phos ERKI 2 1 Low phosp ERK1 2 1 High phos ERK1 2 1 Low phosp ERK1 2 1 High phos ERKI 2 1 Low phosp ERK1 2 1 High phos ERKI 2 1 Low phosp ERKI 2 1 High phos ERKI 2 1 Low phosp ERK1 2 1 High phos ERK1 2 1 Low phosp ERK1 2 1 High phos ERK1 2 1 Low phosp ERK1 2 1 High phos ERKI 2 1 Low phosp ERK1 2 1 High phos ERKI 2 1 Low phosp ERKI 2 1 High phos ERKI 2 1 Low phosp ERK1 2 1 High phos ERKI 2 1 Low phosp ERKI 2 1 High phos ERKI 2 1 Low phosp ERKI 2 1 High phos ERKI 2 1 1 Low phosp ERK1 2 4 mi fe Graph J Image Lane F Bz vop Samples ppErk1 pErk1 Erk1 pERK2 ppERK2 High a HeLa in 5 8 C1 1 Low o HeLa in 5 8 C1 2 Low phospho HeLa in 5 8 C1 4 Chemiluminescen sssggess8 oS a0 Si 32 53 54 55 56 SF S855 GO G4 G2 G2 G4 GS EE G7 EA GS 20 73 pl s EE Capillaries Sample oii Cap Peak Name Position Height Area Area Width 624 0 0239 1538 7 Data for the row s selected in the experiment pane will display as follows Electropherograms in the graph pane display overlaid or stacked depending on the graph option chosen Results for only the selected row s will display in the peaks and capillaries tables The selected row s
22. Opening an Assay To open an existing assay P Pi le Edit Mew Assay Open Assay Save Save s Import Protecal Export Protocol Print Exit Assay folder and select a different assay Compass Software for Peggy User Guide Instrument Window Help Import Template Export Template Select File in the main menu and click Open Assay Protein Sizing Protein Test Peggy Size Simple Western Demo Plate Simple Western Browse Separat gt Separat gt Matrix F Blockin gt Primary r n P Chapter 2 Size Assays A list of the last five assays opened will display Select one of these assays or click Browse to open the Creating a New Assay page 25 Creating a New Assay To create a new assay ProteinSimple recommends using the Peggy template assay and modifying from there as needed Step 1 Open a Template Assay 1 Select File in the main menu and click New Assay T File Edit Instrument Window Help New Assay Peggy Charge Open Assay j Peggy Size Save Save As Import Frotocol Import Template Export Protocol Export Template Print b Exit 2 Alist of template assays that can be used as a starting point for new assays will display Click Peggy Size The Peggy template assay and default settings will display in the Assay screen Compass Software for Peggy User Guide page 26 Chapter 2 Size As
23. Select View in the main menu and click Show Hidden Hidden rows will become visible again in all panes and hidden rows will be marked with an X in the experiment pane E Experiment O a Sample Primary Cycle X Sample Primary Sample Primary Sample Primary Sample Primary Sample Primary X Sample Primary ee 1i Sample Primary e To unhide rows Select the hidden row s Right click on one of the selected rows and click Unhide Setting Run Data Display Filters The filter lets you auto hide specific capillaries in the run file data or only show data for peaks that Compass identified automatically using the user defined peak name analysis parameters NOTE When more than one run file is open filter settings will be applied to all files e To filter data to show specific capillaries only Select View in the main menu and click Filter Uncheck the boxes for the capillaries you do not want shown then click OK Compass Software for Peggy User Guide Viewing Run Data page 293 Fiter Capillaries SSE SSE SS RRA 838888 O oo s CF ka Fe W M y e O Show named peaks only Only data for the checked capillaries will display in data views and results tables unchecked capillaries will be hidden To filter data to show named peaks only Select View in the main menu and click Filter Select Show named peaks only then click OK Only data for peaks that
24. biotin Blocki 2 y hela Blocki 2 E hela Blocki 2 E hela Blocki 2 500 hela Blocki 2 E hela Blocki 2 g 0 hela Blocki 2 c hela Blocki 2 81500 hela Blockin 2 9 hela Blocki 2 hela Blocki 2 E hela Blocki 2 500 biotin Blocki 3 E hela Blocki 3 a hela Blocki 3 8 hela Blocki 3 8 1500 hela Blocki 3 hela Blocki 3 1000 hela Blocki 3 E hela Blocki 3 500 hela Blocki 3 E hela Blocki 3 c 9 hela Blocki 3 8 hela Blocki 3 z 1500 biotin Blocki 4 a hela Blocki 4 1000 hela Blocki 4 E hela Blocki 4 2 509 hela Blocki 4 E hela Blocki 4 hela Blocki 4 o T T T hela Blocki 4 66 90 116 180 hela Rlacki an 4 ot r i 52 B To detach a pane from the main window Click on its tab and drag it outside the main Compass window or right click the tab and click Detached Detached Restore Move Size Minimize Maximize Close To move a detached pane back inside the main window Right click the tab and deselect Detached e Torestore all panes to their original locations Select Window in the main menu and click Default Layout Compass Software for Peggy User Guide page 14 Chapter 1 Getting Started with Compass Restoring the Main Window to the Default Layout To restore screen pane sizes and locations to the original Compass layout select Window from the main menu and click Default La
25. e Tolook at data for one capillary Click a row in the experiment pane Data for just the row selected will display in all data views and tables The following example shows sample data displayed in the lane and peaks panes when one sample is selected jase DemoData_Charge_Hela_ERK12 Compass gt fees File Edit View Instrument Window Help m fg E Assay TE Run Summary fa Experiment o k Graph 2 Image E Lane Ki BF era Sample High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 B aa Capillaries Sample Primary Cap Peak Name Position Height Area Area Width Compass Software for Peggy User Guide page 284 Chapter 9 Charge Assay Data Analysis e Tolook at data for multiple non sequential capillaries Hold the Ctrl key and select the rows you want to view in the experiment pane Data for only the rows selected will display in all data views and results tables The following example shows sample data displayed in the graph in multiple view and capillaries panes when multiple rows are selected DemoData_Charge_Hela_ERK12 Compass e fees File Edit View Instrument Window Help Agia 5 E EB fg E Assay C3 Run Summary
26. hela Block 1 i HH hela Blocki 1 hela Blocki 1 l hela Blocki 1 hela Blocki 1 i 180 hela Blocki 1 l hela Blocki 1 a hela Blocki 1 hela Blocki 1 o biotin Blocki 2 s hela Blocki 2 hela Blocki 2 me Mde 2 O SSS SSSSS Se SS SSS SSS Ss hela Blocki 2 l ERK2 hela Blocki 2 1 MW kDa 42 hela eas Area 36687 hela Blocki 2 hela Blocki 2 1 hela Blocki 2 hela Blocki 2 biotin Blocki 3 ae hela Blocki 3 hela Blocki 3 hela Blocki 3 i hela Blocki 3 4 hela Blocki 3 hela Blocki 3 j hela Blocki 3 i hela AEE Sample Primary Cap Peak Name Position MW kDa Height Area Area Width e Se ee hela Blocki 3 hela Blocki 3 hela Blocki Se eel SS SS Ea a hela Blocki 4 hela Blocki 4 hela Blocki eel ee NE hela Blocki 4 s hela Blocki 4 i hela Blocki Step 5 Checking Samples All immunodetected sample proteins in the graph and lane panes will be labeled automatically with the cal culated protein size Compass Software for Peggy User Guide page 172 Chapter 8 Size Assay Data Analysis NOTE The reported molecular weight for immunodetected sample proteins in Compass may vary slightly from predicted molecular weights based on sample and assay conditions To verify that sample proteins are identified correctly 1 Click the Analysis screen tab 2 Click Show Samples in
27. 15 Click OK to save changes Adding Peak Names Groups 1 Select Edit in the main menu and click Analysis then click Peak Names in the options list 2 Click Add under the analysis settings box A new group will be created Analysis Settings 3 Click on the new group and enter a new name Compass Software for Peggy User Guide Peak Names Settings page 373 Analysis Settings 4 Enter information in the analysis settings peak table as described in Creating a Peak Names Group on page 368 5 Click OK to save changes Modifying a Peak Names Group 1 Select Edit in the main menu and click Analysis then click Peak Names in the options list 2 Click on the group in the analysis settings box you want to modify Analysis Settings 3 Change the information in the analysis settings peak table as described in Creating a Peak Names Group on page 368 4 Click OK to save changes Deleting a Peak Names Group 1 Select Edit in the main menu and click Analysis then click Peak Names in the options list 2 Click on the group in the analysis settings box you want to delete and click Remove Compass Software for Peggy User Guide page 374 Chapter 9 Charge Assay Data Analysis Analysis Settings 3 Click OK to save changes Applying Peak Names Groups to Run Data 1 Select Edit in the main menu and click Analysis then click Peak Names in the options list 2 Click on the group in the analysis
28. 5627 59 33 Clicking the arrow next to a group lists the individual capillaries in the group and reported data for each cap illary Compass Software for Peggy User Guide page 178 Sample 4 SampleA 16 SampleA SampleA SampleA SampleA SampleA SampleA SampleA SampleA SampleA SampleA SampleA SampleA SampleA SampleA SampleA SampleA SampleA 16 SampleA 16 SampleA 16 SampleA 16 SampleA 16 SampleA 16 SampleA 16 SampleB 16 SampleB 16 SampleB 16 SampleB 16 SampleB 16 SampleB 16 SampleB 16 SampleB 16 TO Y F F F F F F F F F F F Y F Primary Primary 1 Primary 1 Primary 1 Primary 1 C2 Primary 1 C Primary 1 Primary 1 Primary 1 Primary 1 Primary 1 C5 Primary 1 Primary 1 Primary1 C6 Primary 1 Primary 1 Primary 1 Primary 1 Frimary 1 Primary 1 Primary 1 Primary 2 Primary 2 Primary 2 Primary 2 Primary 1 Primary 1 Primary 1 Primary 1 Primary 2 Primary 2 Primary 2 Primary 2 ERKI EN 5 RO 15 S 45 u RO 15 v 45 RKO 45 RKO 45 RO 15 ERIN 45 RO 15 RO E A 45 RG REO 45 REO 6 Area 31540 38795 316 0 Std Dev 12487 Name MW kDa fo CV 9 6 Chapter 8 Size Assay Data Analysis The Capillary Groups pane pivots the Peak Groups results to show statistics for named protein peaks in individual columns Sample b SampleA 15 gt SampleA 16 t gt SampleB 16 gt SampleB 16 Grou
29. Cap Gl C11 Ci Ci Gia Gil Peak Name ppErkl pErkl ppERK2 Erk pERK2 Position 499 Data for the row s selected in the experiment pane will display as follows Electrooherograms corresponding to the selected row s are highlighted and displayed in the graph pane Results for selected capillaries display in the peaks and capillaries tables and are highlighted The selected row s are highlighted in the image pane Compass Software for Peggy User Guide Viewing Run Data page 291 IE Graph 2 Image Lane Cycle 1 Samples Exp 240 0 All selected lanes display in the lane pane and lanes corresponding to the selected row s are highlighted E Graph 5 Image F Lane PHB Ae Hiding Capillary Data Capillary data can be hidden from view if needed To do this 1 Click the Experiment tab Compass Software for Peggy User Guide page 292 Chapter 9 Charge Assay Data Analysis 2 Select the rows you want to hide then right click on one of the selected rows and click Hide E Experiment E gt Sample Primary Cycle Sample Primary Sample Primary _ Sample i Primary Sample Primary Sample Primary Sample Primary ries sure 1 Sampie xX Hide Sample H Clear All Data for selected rows will be hidden in all data views and results tables except for the image pane To view hidden rows
30. Change the directory if needed 4 Enter a template name and click Save The template will be saved as a template file Compass Software for Peggy User Guide page 94 Chapter 4 Charge Assays Compass Software for Peggy User Guide page 95 Chapter 5 Running a Charge Assay on Peggy Chapter Overview Starting a Run Stopping a Run Compass Software for Peggy User Guide page 96 Chapter 5 Running a Charge Assay on Peggy Starting a Run Step 1 Get Ready il 2 By 4 6 Open Compass software Prepare instrument empty waste refill water and add a new manifold sponge Create or open desired assay file Prepare assay plate following the procedure described in the product insert IMPORTANT To prevent well evaporation and ensure best results keep a lid on the assay plate until ready to use While plate is spinning add Wash Buffer Anolyte and Catholyte to resource tray cups Place capillary box in the designated resource tray position IMPORTANT Capillaries are light sensitive Keep the cover on the box until you are ready to transfer the capillary box to the resource tray Place assay plate into the sample tray of the instrument and press Start Step 2 Start the Run ile You can start a run in one of two ways depending on what button is displayed in the status bar NEW RUN BUTTON click New Run Compass Software for Peggy User Guide Starting a Run page 97 a Select File in the main men
31. Compass Software for Peggy User Guide Analysis Screen Overview page 267 Edit Menu The following Edit menu options are active View Instrument Windo Cut Copy Ctrl C Paste Ctrl V Analysis Preferences Copy Lets you copy data shown in the graph lane peaks or capillaries panes See Copying Data Views and Results Tables on page 310 for more information Analysis Displays the analysis settings used to analyze the run data and lets you change them as needed See Compass Analysis Settings Overview on page 348 for more information Preferences Lets you set and save custom preferences for data export plot colors in the graph and Peggy s Twitter settings See Chapter 10 Setting Custom User Preferences for more information View Menu The following View menu options are active Instrument Window Single View Multiple View Standards Registration Samples Filter View Region Show Hidden Single View Displays data in a per capillary single view format e Multiple View Displays data in a per 12 capillary multiple view format Standards Lets you change the data view to show only the fluorescent standards Registration Lets you change the data view to show only the capillary registrations Samples Lets you change the data view to show only immunodetected sample proteins e Filter Lets you display data only for specific capillaries or named proteins View Regi
32. Compass Software for Peggy User Guide Standards Settings Standards Settings page 249 The standards analysis settings page lets you view and change the molecular weight and position for ladder and fluorescent standards set the registration peak and change the capillary used for the ladder To access these settings select Edit in the main menu and click Analysis then click Standards in the options list NOTE Settings can be modified in an assay prior to starting a run or in a run file once the run has finished executing Analysis changes made to an executing run will not be saved to the final run file w snr snpewenenencoene NN A Advanced Images Peak Fit Peak Narnes Standards Analysis Settings Standard Fluorescent Peaks MW Position Fit Registration 1 100 J 12 200 P 180 90 MH O Cren Cremo Override Ladder Capillary i gt MW Position 12 200 40 500 66 600 700 800 900 90 116 180 KIER EEE a a eei ai ras Restore Original Click Import to import an analysis settings file This will be explained in more detail in Importing Analysis Settings on page 261 Compass Software for Peggy User Guide page 250 Chapter 8 Size Assay Data Analysis Click Export to export the current analysis settings file This will be explained in more detail in Exporting Analysis Settings on page 261 Click Restore Original to restore Compass default sett
33. Cycles 1 through 3 capillaries 6 8 and 10 Cancel Compass Software for Peggy User Guide Peak Names Settings page 247 6 Ifyou need to change the peak names group used for a data set click the cell in the Settings column and click the down arrow Select a group from the drop down list Apply Settings Settings ERKL 2 m ERK1 2 a STAT 7 Repeat the previous steps to apply other groups to specific run data 8 To remove a data set click on its cell in the Apply To column then click Remove Compass Software for Peggy User Guide page 248 Chapter 8 Size Assay Data Analysis 9 Click OK to save changes Named peaks will be identified with a peak name label in the electrophero gram and virtual blot and will be color coded in the peaks and capillaries tables ya adel E Assay T Run Summary EE Analysis Chemiluminescence SoQSBRBUSRSSSRSRSAS RSS PLCg v ERK STATS Biotin v ERK STATS Primar PLCg Primar v ERK Primar STATS Primar PLCg Primar v ERK Primar STATS Primar PLCg Primar v ERK Primar STATS Primar Biotin Primar v ERK Primar STATS Primar PLCg Primar vV ERK Primar STATS Primar PLCg Primar 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 Sample Primary Cap Peak Name Position MW kDa Height Area Width S N PLCg Primar 33 2 cf F amp F FFF Few hHw ww ww ew
34. KIOOOE K A A EE 7 Enter the position of the fluorescent standard peak New Standards Fluorescent Peaks Registration ca N N EIEIEI EE Compass Software for Peggy User Guide page 381 page 382 Chapter 9 Charge Assay Data Analysis NOTE Standards peak positions are relative to each other Only the difference in their position is used to help identify the standard peaks When entering standard peak information for the first time review the standards data in the Analysis screen to find the correct peak position 8 Repeat the steps above for the remaining standards in the table To add another standard Click Add under the peak table then modify the information in the new row Toremove a standard Select its row and click Remove 9 Select which standard should be used for capillary registration by clicking the checkbox in the Registra tion column The first and last standards are typically used for the registration New Standards Fluorescent Peaks Position Registration Fi 210 350 520 550 650 KIOOOE NOTE In order for Compass to perform data analysis at least one peak must be selected for registration 10 Select which standards should be used for pl determination of sample proteins by clicking the check box in the Fit column The standards not used for registration are typically also used for fit New Standards Fluorescent Peaks Position Registration 210 350 520
35. Lets you change the data view to show only the fluorescent standards Registration Lets you change the data view to show only the capillary registrations Samples Lets you change the data view to show only immunodetected sample proteins e Filter Lets you display data only for specific capillaries or named proteins View Region Lets you change the molecular weight x axis range of the data displayed Show Hidden Shows capillaries that are hidden from the data view Compass Software for Peggy User Guide page 136 Opening Run Files Chapter 8 Size Assay Data Analysis You can open one or multiple run files at a time to compare data between runs Opening One Run File NOTE If you need to open a run file when another run is executing launch another instance of Compass first then open the file 1 Select File in the main menu and click Open Run Edit Instrument Window Help Open Run b Add Run gt Close Close All Exit OHS STS SAAT O Sworn prt Instrument Sally Sally Started Wed 8 20 F Completed Thu 2 57 Pt Sally NFkB pathway test 2 Sally NFkB pathway test 1 Sally HeLa IKb test 2012 03 05_11 51 19_HelaControlERKassay 2012 03 05_14 45 07_2012Mar05Multi Western_IkB 2012 03 14_HeLa_ERK STAT3 PLCg_Day5_PLO003 Copy Sally HeLa Day 3 Run Sally HeLa TNF treatment Sally MCF7 Multi target screen 2012 03 12_MCF7sc_PBK p110b cs3011_filtAD_PLOO3 Browse 2 Alist of the last 10 runs
36. Open the assay you want to export the assay protocol from 2 Select File in the main menu and click Export Protocol The following window displays Owe di Documents My Documents Compass gt Assays Search Assays Organize v New folder wr Favorites Documents library z Desktop Assays m3 Downloads T i Recent Places Arrange by Folder Name Date modified Type _ Standard sample protocol protocol 10 4 2011 9 57 PM PROTOCOL File BB Desktop Libraries E Documents E My Documents di Compass 1 File name New protol ge Save as type Protocol File protocol z a Hide Folders 3 The default directory is Compass Assays Change the directory if needed 4 Enter a protocol name and click Save The protocol will be saved as a protocol file Importing an Assay Template NOTE Importing an assay template imports information into the Template pane only 1 Open the assay you want to import the assay template in to 2 Select File in the main menu and click Import Template 3 Select a template file template and click OK The imported information will display in the Template pane Compass Software for Peggy User Guide page 50 Chapter 2 Size Assays Exporting an Assay Template NOTE Exporting an assay template exports information in the Template pane only 1 Open the assay you want to export the assay template from 2 Select File in the main menu and click Exp
37. Position Height 87 3 3 386 1 31 If standards are not identified correctly they can be manually corrected as follows If an incorrect band is identified as a standard Right click the band in the lane or peaks table and select Not a Standard Compass should correctly reassign the remaining bands as Standards e To set an unidentified band as a standard Right click the band in the lane or peaks table and select Force Standard Compass will assign the band as a standard and correctly reassign the remaining standard bands c Repeat the previous steps for the remaining rows in the experiment pane to make sure all standards are identified correctly Compass Software for Peggy User Guide page 168 Step 3 Checking Capillary Registrations All capillaries should have a single capillary registration peak To verify the registrations are identified cor rectly 1 Click the Analysis screen tab Chapter 8 Size Assay Data Analysis Click Show Registrations and Single View in the View bar 2 3 Click the Graph tab 4 Click on the first row in the experiment pane The registration peak will be the first and largest peak in the electropherogram Check that the registration peak is identified and labeled Reg 1 in the electro pherogram It will also be identified with a purple R in the peaks table File Edit View Inst ment Window Help 0 ts Assay C3 Run Summary 6 A so Sa
38. Remove Restore Original Compass Software for Peggy User Guide page 222 Chapter 8 Size Assay Data Analysis Click Import to import an analysis settings file This will be explained in more detail in Importing Analysis Settings on page 261 Click Export to export the current analysis settings file This will be explained in more detail in Exporting Analysis Settings on page 261 Click Restore Original to restore Compass default settings Click OK to save changes and exit Click Cancel to exit without saving changes Standards Settings Peak Width The approximate width at full width half max used to filter out fluorescence artifacts which improves recognition of standards The default value is 15 Allowable Drift The distance in pixels that standards are expected to move compared to their entered positions in the standards analysis page This setting assists in recognition of standards The default value is 100 Sample Settings Peak Fit Starting Width Ratio Focuses the peak fit towards the peak center and aids the overall peak fitting The default value is 0 5 Image Settings Median Filter Threshold Ratio Pixel ratio used to filter out camera artifacts The default value is 0 5 Median Filter Threshold Limit Pixel threshold value used to filter out camera artifacts The default value is 100 Compass Software for Peggy User Guide Advanced Analysis Settings page 223 Advanced Analysis Settin
39. Step 2 SGM WCU nhs aap ceah A 96 Step POSTRUN FMOCCOUICS oarre rrr EEE 103 OPONGA U erria a ee RA 104 Chapter 6 Run Status a 105 RU SUMINMNIGIY S TE T OVON OW eian EEEIEI 106 RUT SUNTAN SCEN FANES arere 106 Software Menus Active in the Run Summary eaaa eRe EE E et ee EEEE 107 OD CARING RUN FES oc anrr AN 109 OPENT ONERU HC irc ok T ceceenares a 109 Opening Multiple Run Files a na 109 Viewing File and Run Status Information ee Assay Steps Size based ASSAYS n 06 00 econ 12 Assay Steps Charge based ASSAYS 1713 Watching Standards Separation Movies 1715 Viewing Current and Voltage Plots 04 7 Switching Between Open Run Files 00 08 118 CONG Oa E E EE 118 Chapter 7 Controlling Peggy 119 HEN GOO e aa a EN 120 OOTO OAN WA U een E a AEE iA 120 DCN ITOS spea a ER 120 ICOMOS EEEE A EA E EEE 21 Dele E E NEE EA E T 124 Viewing and Changing System Properties 124 VENLO E AEREE EA OREA 25 EO N Sane Meee 125 DC RES e ETES EET TT 128 Compass Software for Peggy User Guide page iii B E E OT E E EET 130 Chapter 8 Size Assay Data Analysis 131 BNGIVSIS Sereen WENE W sen santas oa dtontpeeaiepenae s 132 ANANS OTEN PANE o s oot N 32 Software Menus Active in the Analysis Screen 134 OCI RUFE rs ste d ELERO RERS 136 Opening One RUFIE os cers rotra EE 136 Opening MUITIDIC RUN FCS erori To EGAS 136
40. are highlighted in the image pane Compass Software for Peggy User Guide Viewing Run Data page 289 og Graph 2 Image Lane Cycle 1 Samples Exp 240 0 Lanes for only the selected row s are displayed in the lane pane E Graph 5 Image 5 Lane PMB DA b b RY RX S oe x Y x WF SW x se g 3 Se a T ae Ss Q YS ws Ss Compass Software for Peggy User Guide page 290 Chapter 9 Charge Assay Data Analysis To view data up to 96 capillary format Click Multiple View in the View bar or select View in the main menu and click Multiple View DemoData_Charge_HeLa_ERK12 Compass File Edit View Instrument Window Help me Assay T Run Summary Ta Experiment A Sample High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp q ilies u t nse g m A z ee ww lt gt Image J Lane Tf 7 0 Chermibaminescence Chemibminescence Chemibminescence Chemibmninescenice E Peaks t Capillaries
41. enable Access Control by clicking Edit then Preferences and Access Control Select Enable close Compass then re launch Compass with a valid log in Uncontrolled file cannot be opened when logged in To open an uncontrolled Run file disable Access Control by clicking Edit then Preferences and Access Control Deselect Enable close Com pass then re launch the software NOTE Uncontrolled Assay files can be opened when Compass Access Control is enabled controlled mode Command disabled Certain commands are only available when a user with the correct permissions is logged in To change user permissions use a web browser to log in to the Authorization server web interface at the address shown on the Access Control page in Preferences such as 10 1 3 231 8000 Compass does not prompt for log in Compass will only prompt for a log in on launch when Access Control is enabled in Preferences Enable Access Control by clicking Edit then Preferences and Access Control Select Enable close Compass then re launch the software You should now be prompted for a log in Authorization Server The Authorization Server controls the log in access to Compass In the simplest configuration the server is run on the same computer as Compass and only that copy of Compass is controlled A single server can also be used to control access to multiple copies of Compass running on different computers so long as they have network access to the server Mu
42. ja ca ee C a cn eae _ K oo _ ca EE n e Led eS SS i oea m a E es e 2 Click the Overlay Standards Data button again to return to the default auto aligned view Compass Software for Peggy User Guide page 324 Chapter 9 Charge Assay Data Analysis Moving Lanes in the Virtual Blot View Lanes in the virtual blot view can be moved to make it easier to compare specific lanes of data To do this 1 Click a lane in the virtual blot Hold the mouse button down and drag the lane to a new position fz Graph ka Image E E Ba VALLES Mhdipiiidsdidiiddd dd oF ot oF FM FAS SPCC PEPE SESE 2 Release the mouse button The lane will now be repositioned in the virtual blot view Compass Software for Peggy User Guide Changing the Electropherogram View page 325 Changing the Electropherogram View Options in the graph pane let users zoom and scale electropherograms overlay or stack plots and change the peak and plot information displayed The graph pane toolbar has the following options Auto Scale jain Graph Options Stack the Plots Overlay the Plots Compass Software for Peggy User Guide page 326 Chapter 9 Charge Assay Data Analysis Autoscaling the Electropherogram Click the Autoscale button to scale the y axis to the largest peak in the electrooherogram ppErk1 pErk1 Erk ppERK Low phospho
43. then paste into another document File Edit Instrument Window Help Et Assay C Run Summary AE Analysis Run 2013 04 15_09 49 28 Simon_short Separation _ lt IV Plot YY Status History Date User Name Message Comment 04 15 2013 9 48 AM b RunStart2013 04 15_ 09 49 28 Simon_short 05 10 2013 4 22 PM steveg Saved analysis changes Changed Baseline fr 05 10 2013 4 44 PM steveg e Signature applied Approved lt Time Message Comment Steven Gallagher steveg is logged in Troubleshooting Problems and Suggested Solutions If any of the following error messages are encountered follow the recommended steps below to resolve the issue Unknown user name or password Check if the Caps Lock is on user name and password are case sensitive Aska Compass administrator to confirm your user name If your password is unknown then the administrator can reset your password see Resetting User Passwords on page 418 for more information Compass Software for Peggy User Guide page 410 Chapter 11 Compass Access Control and 21 CFR Part 11 Compliance Server not available From the Edit menu click Preferences and then Access Control to confirm the server address is set to the correct Authorization server address Compass must be able to reach the server on the network e The server must have inbound access to port 8000 enabled Controlled file cannot be opened without log in To open a controlled Run file
44. 120 0 120 0 120 0 gt Secondary Ab Time min 60 0 60 0 60 0 60 0 60 0 60 0 60 0 60 0 gt Detection Each row contains an assay protocol step Each step can contain a reagent assay plate row assignment and one or more parameter settings To view the details for a step click on the white arrow next to the step name ProteinSimple recommends using the default protocol settings for Peggy assays An expanded list of the default protocol step parameters is shown Compass Software for Peggy User Guide Creating a New Assay Separation Matrix Well Row Load Time sec 4 Stacking Matrix Well Row Load Time sec Sample 07 12 0 12 0 12 0 12 0 07 12 0 07 12 0 07 12 0 07 12 0 Well Row Set ES et EE EE Es ean ees 8 0 8 0 8 0 8 0 8 0 8 0 8 0 8 0 Load Time sec Separation Time min Separation Voltage volts Standards Exposure sec Immobilization Time sec Matrix Removal Matrix Removal Time sec Matrix Washes Matrix Wash Soak Time sec Wash Soak Time sec Blocking Time min Well Row Washes Wash Soak Time sec Primary Ab Time min 40 0 250 5 0 200 0 140 0 3 150 0 150 0 23 0 150 0 120 0 40 0 250 5 0 200 0 150 0 120 0 40 0 250 5 0 200 0 140 0 3 150 0 150 0 23 0 150 0 120 0 40 0 250 5 0 200 0 150 0 120 0 40 0 250 5 0 200 0 40 0 250 5 0 200 0 40 0 250 5 0 200 0 Well Row Sa Se Se eS ee ese SE ae 2 2 2 2 2 2 2 2 Washes Wash
45. 194 Chapter 8 Size Assay Data Analysis a a Fi Overlay Standards Data Po j a E E ee E Ee E 2 Click the Overlay Standards Data button again to return to the default auto aligned view Compass Software for Peggy User Guide Changing the Virtual Blot View page 195 Moving Lanes in the Virtual Blot View Lanes in the virtual blot view can be moved to make it easier to compare specific lanes of data To do this 1 Click a lane in the virtual blot Hold the mouse button down and drag the lane to a new position ee Graph Le Image E ane gt OK Be 2 Release the mouse button The lane will now be repositioned in the virtual blot view Compass Software for Peggy User Guide page 196 Chapter 8 Size Assay Data Analysis Changing the Electropherogram View Options in the graph pane let users zoom and scale electropherograms overlay or stack plots and change the peak and plot information displayed The graph pane toolbar has the following options Auto Scale BF Graph Options Stack the Plots Overlay the Plots Compass Software for Peggy User Guide Changing the Electropherogram View page 197 Autoscaling the Electropherogram Click the Autoscale button to scale the y axis to the largest peak in the electrooherogram Gnas EH ane Trga s5 5s SgsSSg Chemiluminescence z 5500 5000 4500 4000 3500 3000 2500 chemiluminescence Compass S
46. 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERE1 2 Primary 1 ERKL 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERE1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 1 ERK1 2 Primary 2 ERK1 2 Primary 2 ERK1 2 Primary 2 ERK1 2 Primary 2 rhea MI i 4 page 307 The Capillary Groups pane pivots the Peak Groups results to show statistics for named protein peaks in individual columns b Hi phospho HeLa 24 b Hi phospho HeLa 24 b Low phospho HeLa 24 ERK1 2 Primary 1 b Low phospho HeLa 24 ERK1 2 Primary 2 lal Group Plot Primary Capillary ppErkl ERK1 2 Primary 1 ERK1 2 Primary 2 Compass Software for Peggy User Guide Std Dev pErkl Std Dev CV Erki Std Dev CV SEM page 308 Chapter 9 Charge Assay Data Analysis Group Plots The mean values for named peaks in each group are plotted in bar graphs with error bars showing the stan dard deviation The plots compare different antibodies for the same sample and different samples for the same antibody to allow a choice of presentation a Peak Groups eH Capillary Groups Hi phospho HeLa Run 1 Low phospho HeLa Run 1 S Average Area Average Area L 2 aw ot wo ow ERK1 2 Primary 1 Run 1 ERK1 2 Primary 2 Run 1 Average Area Hiding or Removing Capillaries in Group Analysis Hidden capillaries are not included in groups However hiding capillaries provides an e
47. 299 r DemoData_Charge_Hela_ERK12 Compass o File Edit View Instrument Window Help vEt z2 mw i fg E Assay T Run Summary Ea Experiment u E Graph te Image Lane gt D i BF a o Sample Primary Cycle Ra Ra Ra High phos ERK1 2 1 ee F a o e s Low phosp ERKI 2 1 High phos ERKI 2 1 Low phosp ERKI 2 1 High phos ERKI 2 1 Low phosp ERKI 2 1 High phos ERKI 2 1 Low phosp ERKI 2 1 High phos ERKI 2 1 Low phosp ERKLI 2 1 High phos ERKI 2 1 Low phosp ERKI 2 1 H Peaks N FE Capillaries Bs Sample Primary Cap Peak Position Height a High ERK1 2 C1 1 1 16 40 6 a High ERK1 2 C11 2 72 15 6 High ERK1 2 Cll 3 84 16 1 High ERK1 2 C11 4 97 12 8 High ERK1 2 C11 5 130 113 High ERK1 2 C1 1 6 261 12 0 High ERK1 2 C1 1 7 271 10 9 _ S Hinh _FRK1 2 aa R 314 202 4 x If standards are not identified correctly they can be manually corrected as follows Ifan incorrect band is identified as a standard Right click the band in the lane or peaks table and select Not a Standard Compass should correctly reassign the remaining bands as standards To set an unidentified band as a standard Right click the band in the lane or peaks table and select Force Standard Compass will assign the band as a standard and correctly reassign the remaining standard bands c Repeat the previous steps for the remain
48. 55 DO 005 10 Click in the first cell in the Range column Compass Software for Peggy User Guide Peak Names Settings page 371 Analysts Settings ERKI Name pl Color Range Show perk 55 E E 11 Enter a range window for the pl entered Compass will automatically name peaks found within this per cent of the pl For example if the pl entered is 5 5 and a 0 1 pl range is used all peaks between pl 5 4 and 5 6 will be identified with this peak name NOTE The reported pl for immunodetected sample proteins in Compass may vary slightly from predicted pls based on sample buffer and assay conditions 12 Select the checkbox in the first cell of the Show column This will turn peak naming on for the sample protein Analysis Settings ERKI Name pl Color Range Show ppErd 55 a o To turn peak naming off for a particular sample protein deselect the checkbox in the Show column 13 To add another sample protein click Add under the analysis settings peak table Analysis Settings ERKI Name pl Color Range Show ppErkl 55 ae 0 Peak 6 ooo nana Compass Software for Peggy User Guide page 372 Chapter 9 Charge Assay Data Analysis 14 Repeat the previous steps to enter information for other sample proteins In the following example three sample proteins were entered Analysis Settings ERKI pl Color Range Show 55 E 575 B oos 59 E To remove a sample protein select its row and click Remove
49. 8 2012 11 05 AM Safari Document 36 KB jr Compass for NanoPro 1000 pdf 10 8 2012 10 56 AM Adobe Acrobat D 6 002 KB gt i configuration H Compass for Peggy pdf 10 8 2012 10 56 AM Adobe Acrobat D 7 184 KB gt ig Examples FE Compass for Sally pdf 10 8 2012 10 56 AM Adobe Acrobat D 7 184 KB gt features E Compass for Simon pdf 10 8 2012 10 56 AM Adobe Acrobat D 5 191 KB gt ge jre Compass exe 10 8 2012 11 04 AM Application 52 KB p2 Compass ico 10 8 2012 11 07 AM Icon 24 KB gt ig plugins E Compass ini 10 8 2012 11 05 AM Configuration sett 1KB readme compass_data_file ico 10 8 2012 11 07 AM Icon 25 KB gt Gm templates E eclipsec exe 10 8 2012 11 04 AM Application 24 KB epl v10 html 2 25 2005 6 53 PM HTML Document 17 KB license rtf 10 8 2012 10 56 AM Rich Text Format 139 KB NanoPro 1000 User Guide pdf 10 8 2012 10 56 AM Adobe Acrobat D 6 002 KB notice html 2 4 2011 3 39 PM HTML Document 9KB TE Peggy User Guide pdf 10 8 2012 10 56 AM Adobe Acrobat D 3 741 KB FE Sally User Guide pdf 10 8 2012 10 56 AM Adobe Acrobat D 3 741 KB ap Simon User Guide pdf 10 8 2012 10 56 AM Adobe Acrobat D 1 082 KB Compass Software for Peggy User Guide Directory and File Information page 17 Compass assay and run files are located in the Documents folder in the User directory File Edit View Tools Help Organize v Share with v Burn New folder a vir Favorites Documents library MD Desktop Compass se Downloads
50. 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 26 019 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 31 035 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 36 049 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 41 062 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 46 077 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 51 093 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 56 110 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 01 128 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 06 146 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 11 163 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 16 180 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 21 197 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 26 215 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 31 232 tem
51. CGI PIE GlOUD creperie n 364 Applying Peak Fit Groups to Specific Run Data 364 POGK NOTES SCUHINOS ice ccusmtiots wheat cduatce cess 36 7 Peak Names Analysis Settings Groups 368 Creating a Peak Names Group 065 368 Adding Peak Names GrOUDS veecrsrcsicireiiaa 372 Modifying a Peak Names Group 64 573 Deleting a Peak Names Group 6 005 373 Applying Peak Names Groups to Run Data 374 SUONGOIOS SOMINGS ei a 378 Standards Analysis Settings Groups 379 Creating a New Standards Group 380 Changing the Default Standards Group 383 MOGINVING G STGNG GIGS GOUD treser aa 383 Deleting an Analysis GrOUD ersi eirinn is 384 Applying Analysis Groups to Specific Run Data 384 Importing and Exporting Analysis Settings 586 Importing Analysis Settings 0 000 386 EDONG ANGIVS S SE UNGS aa a naeia 387 Chapter 10 Setting Custom User Preferences 389 Custom Preference OPtions 0 00 c ccc e cece 390 SEUNG DOUTE POIT OVLON va ccctscnraeeeteak ences 39 Selecting Custom Plot Colors for Graph Overlay 392 Setting Peggy Up to Send Tweets 00 cee 394 Chapter 11 Compass Access Control and 21 CFR Part 11 Compliance 401 VOIMICW EEEE I E EEE TETEE EE 402 aog ACES CONTO ar a Bastin 403 LOGGING ITO CONDOS riat AEE E 404 RESOIVING LOG IN ISSUCS eiei ia a aai 405 SAVNO C NON GE Sre r AALA Ai 405 Ea T FN E E EE O TE 406 HOS
52. Compass Software for Peggy User Guide page 234 Chapter 8 Size Assay Data Analysis Analysis Settings Peak Fit STAT peak fit 4 Modify range baseline or peak find parameters as needed 5 To use the new group as the default peak fit settings for the run file data click the arrow in the drop down list next to Default then click the new group from the list Peak fit settings in the new group will then be applied to the run data Analysis Settings Peak Fit STAT peak fit Peak Fit Override STAT peak fit 6 Click OK to save changes Changing the Default Peak Fit Group 1 Select Edit in the main menu and click Analysis then click Peak Fit in the options list 2 Click the arrow in the drop down list next to Default then click a new default group from the list Analysis Settings Peak Fit STAT peak fit Default Peak Fit Override STAT peak fit Compass Software for Peggy User Guide Peak Fit Analysis Settings page 235 3 Click OK to save changes Peak fit settings in the group selected will be applied to the run data Modifying a Peak Fit Group 1 Select Edit in the main menu and click Analysis then click Peak Fit in the options list 2 Click on the group in the analysis settings box you want to modify Analysis Settings Peak Fit STAT peak fit 3 Modify range baseline or peak find parameters as needed 4 Click OK to save changes The new peak fit settings will be applied to th
53. Copying Data Views and Results Tables You can copy and paste data and results tables into other documents or save a data view as a graphic file Copying Data Views 1 Click in the graph or lane pane 2 Select Edit in the main menu and click Copy or right click and select Copy 3 If you selected copy from the graph pane the following window will display click Copy If you selected copy from the lane pane skip to the next step Graph title Samples Metafile EMF Bitmap PNG Portable Document Format PDF Save Copy _ Cancel i 4 Open a document Word Excel PowerPoint etc Right click in the document and select Paste A graphic of the copied data view will be pasted into the document Copying Results Tables 1 Click in the peaks or capillaries pane 2 Select one or multiple rows 3 Select Edit in the main menu and click Copy or right click and select Copy A Open a document Word Excel PowerPoint etc Right click in the document and select Paste Data for the rows selected will be pasted into the document Saving the Graph View as an Image File 1 Click in the graph pane 2 Select Edit in the main menu and click Copy or right click and select Copy 3 Select an image option EMF PNG or PDF in the pop up window then click Save Compass Software for Peggy User Guide page 182 Chapter 8 Size Assay Data Analysis Graph title Samples 5 Metafile EMF Bitmap PNG E
54. Fit Baseline Corrected and Sample are selected the fit plot is on the bottom lea Graph a i Image Lane EF B m K562 C1 10 40 MW kDa NOTE When viewing standards or registration data this option is called Standards Fluorescence or Regis tration Fluorescence respectively Compass Software for Peggy User Guide page 216 Chapter 8 Size Assay Data Analysis Standards Checking this box aligns the molecular weight of the raw standards data to the sample data and overlays both electropherograms in the graph pane a Image Lane 28 Bs 5 77m Samples kn Graph 28228 38 shemilumines cence MW kDa Adding and Removing Baseline Points Points in the baseline can be added or removed as needed To do this 1 Click the Graph Options button in the graph pane toolbar and check Baseline Fit This will display baseline points for the raw sample data 2 Use the mouse to draw a box around the area you want to correct This will zoom in on the area Compass Software for Peggy User Guide Changing the Electropherogram View page 217 3 Right click a baseline point and click Add Baseline Point or Remove Baseline Point An raph gt Image Lane a Be a z Samples Zoom Out Add Peak Add Baseline Point oe Remove Baseline Point FE Peaks HE Capillaries Clear All m Sample Primary Cap Peak Name Position MW k Copy Ctrl C Width S
55. Group across runs Group across cycles Groups capillaries run in different cycles Group across runs Groups capillaries from multiple runs This option creates fewer groups and generates statistics across multiple runs No option selected When only one run is open groups will only contain capillaries from the same cycle When more than one run is open groups are created for each run Compass Software for Peggy User Guide Group Statistics Viewing Statistics Peak and Capillary Groups page 177 The Peak Groups pane reports statistics for each named protein in every group Each group shows the statis tics for named proteins which includes average area standard deviation and CV The number in parenthe sis after the sample name indicates the number of capillaries in the group b b D D D D D D D D D D D P SampleA 16 SampleA 16 SampleA 16 SampleA 16 SampleA 16 Sample 16 SampleA 16 Sample 16 SampleB 16 SampleB 16 SampleB 16 SampleB 16 SampleB 16 SampleB 16 SampleB 16 SampleB 16 Primary Cap Primary 1 Primary 1 Primary 1 Primary 1 Primary 2 Primary 2 Primary 2 Primary 2 Primary 1 Primary 1 Primary 1 Primary 1 Primary 2 Primary 2 Primary 2 Primary 2 EE Group Plot 3 Bia ea MW kDa Area 31540 23175 95703 44543 27978 20482 4974 39586 32337 23464 96765 44822 30272 22405 90564 42179 Std Dev CV 18796 Bm2
56. HeLa C1 12 1 TI D TI Liri D E 3 E D O 200 650 200 750 700 650 600 10 500 450 400 ao 300 2a 200 150 100 30 50 51 52 53 54 55 56 57 58 59 60 G1 62 63 64 65 66 amp 7 GB 69 70 pl Click the Autoscale button again to return to default scaling ppErk1 pErk1 Erk1 ppERK2 2588588388828868 u TI E T TI Fa a E E 3 E T Lo s0 64 52 53 54 55 56 57 SE 59 EN Ki pl Compass Software for Peggy User Guide Changing the Electropherogram View page 327 Stacking Multiple Electropherograms Electropherogram data for multiple capillaries can be stacked vertically in the graph pane for comparison To do this 1 Click Single View 2 Select multiple rows in the experiment pane 3 Click the Stack the Plots button The individual electrooherograms for each row selected will stack in the graph pane kn Graph Image Lane EB Ed e Samples Stack the plots 15007 ppErk1 pErk1 Erk1 pERK2 ERK 1000 ppERK Hi phospho HeLa L 1U Hi phospho HeLa C1 1 Low phospho HeLa 500 s 000 500 si 000 a C110 5 m 000 500 s 000 500 0 Hi phospho HeLa C5 5 Low phospho HeLa C5 6 ChemilumineS hen uMines hentitumines hen suMineehemaumines cence ni si s2 s3 s4 5a aE a7 oE S9 EU EI cA cg ed Es eE E7 CR bg A pl Users can customize the colors used for the stacked plot display For more information see Selecting Cus tom Plot Colors for Graph
57. Layout a XOB1 234 Lj Separation Me Insert a luminol row Separation Tir Separation Vo Matrix Remow A Ja B B E Blocking Time D D E E Primary Ab Tii F F a a Secondary Ab z z Detection J J K K To insert a fourth or fifth incubation reagent Click an empty row or the row below where the new incubation reagent should be inserted then click Insert a 4th incubation step 4 icon in the Layout pane toolbar A new incubation reagent row will be added in the empty row or inserted above the selected row Compass Software for Peggy User Guide Creating a New Assay page 29 E Layout x OB 2 34 La la Separat Matrix F Blockin Primary Second Tertiary Detecti see TaAmMmMonaE Ze rOn OOP A fifth incubation reagent can now be added by repeating the above and clicking Insert a 5th incu bation step 5 icon instead NOTES When using the fourth and fifth incubation steps only five cycles can typically be performed per run as more water is needed per cycle for the additional wash steps Row J is the last row assignment that can be used on the assay plate New sample incubation or luminol rows cannot be inserted if row J already has an assigned reagent To insert a row in this case you must first move the contents of row J to another area on the plate To delete a row Click the row to be deleted then click Delete red x icon in the toolbar Only empty rows and fourth i
58. Load Time sec 20 0 Wash Soak Time sec 150 0 Tertiary Ab Time min 10 0 Detection NOTE Only rows designated as secondary antibody in the Layout tab can be selected in the Well Row drop down menu 7 Additional exposures can be collected in the assay if desired To do this click the white arrow next to Detection to expand the row Click the cell in the exposure column next to Detection Profile to open the Detection Profile window Additional times can be added to the protocol by clicking the Add button entering the values and selecting OK Compass Software for Peggy User Guide page 76 Chapter 4 Charge Assays Protocol TE Notes o O Add Remowe Sample Separation Immobilization Time sec 100 0 Primary Ab Time rin 120 0 Secondary Ab Time min 60 0 Tertiary Ab Time rnin 10 0 Detection Well Row El Wash Load Time sec 2 0 Detection Profile 5 Exposures 8 Modify any other protocol parameters as needed 9 To add additional cycles to the assay protocol click in any cell with a value in a cycle column To add one cycle either click Add or click the down arrow next to Add and select 1 Cycle Select 4 Cycles from the drop down menu to add four additional cycles Select All Cycles from the drop down menu to add the number of cycles needed to reach the maximum of eight Add Remove 1 Cycle Sample 4 Cycles Separation Immobilization Time sec All Cycles Primary Ab Time rnin
59. Name Date modified Type Size S Recent Places Arrange by Folder Assays 10 17 2012 6 33 PM File folder 4 I Desktop i New Assays 10 11 2012 12 07 PM File folder nr f Y 4 Lead Runs 10 11 2012 12 06 PM File folder E3 Documents S E My Documents 4 Compass Assays di New Assays Runs Assays Folder Contains all saved assay files New Assays Folder Contains assay template files Runs Folder Contains all run files Run data is automatically written to this folder NOTE When a Compass software update is performed the template assays in the New Assays folder are overwritten If you have customized these assays ProteinSimple recommends saving them in a unique subfolder prior to updating the software then transferring them back to the New Assays folder after the update to avoid losing your assay customizations Compass Software for Peggy User Guide page 18 Chapter 1 Getting Started with Compass File Types The following file types are used by Compass Assay Files Use an assay file extension Run Files Use a cbz file extension The default file format for run files is Date_Time_AssayName An example run file name would be 2012 09 28_18 50 53_ Simple Western cbz Protocol Files Exported protocol files use a protocol file extension Template Files Exported template files use a template file extension Analysis Settings Files Exported analysis settings files use a settings file e
60. Open Assay k Save Save As Import Protecal Import Template Export Protocol Export Template Print p Exit New Assay Creates a new assay from a starter template Open Assay Opens an existing assay Save Saves the open assay Save As Saves the open assay under a different file name Import Protocol Imports a saved protocol file into an assay Import Template Imports a saved template file into an assay Export Protocol Exports the current protocol file for future use Export Template Exports the current template file for future use Print Prints the information in the Protocol or Template panes Exit Closes Compass Compass Software for Peggy User Guide page 64 Chapter 4 Charge Assays Edit Menu The following Edit menu options are active Instrument Window Help Cut Copy Ctrl C Paste Ctrl V Default Analysis Analysis Preferences Copy Copies the information in the Protocol or Template panes into other documents Default Analysis Displays the default settings that will be used to analyze the run data generated with an assay Analysis Not active in this screen Preferences Set and save custom preferences for data export plot colors in the graph and Peggy s Twitter settings See Chapter 10 Setting Custom User Preferences for more information Compass Software for Peggy User Guide Reagent Color Coding page 65 Reagent Color Coding The Assay scree
61. Position 5 m 45 515 70 615 100 T15 120 815 195 920 16 To use the new group as the default analysis settings for the run file data click the arrow in the drop down list next to Default then click the new group from the list Analysis settings in the new group will then be applied to the run data Analysis Settings Standard New Standards Default j ae E Standard New Standards 17 Click OK to save changes Changing the Default Standards Group 1 Select Edit in the main menu and click Analysis then click Standards in the options list 2 Click the arrow in the drop down list next to Default then click a new default group from the list Compass Software for Peggy User Guide page 258 Chapter 8 Size Assay Data Analysis Analysis Settings Standard New Standards Default aaah as x Standard Mew Standards 3 Click OK to save changes Analysis settings in the group selected will be applied to the run data Modifying a Standards Group 1 Select Edit in the main menu and click Analysis then click Standards in the options list 2 Click on the group in the analysis settings box you want to modify Analysis Settings Standards New Standards 3 Modify fluorescent standards and ladder standards information as described in Creating a New Stan dards Group on page 252 4 Click OK to save changes The new analysis settings will be applied to the run data Deleti
62. Position Registration HoOoos ES Default Std Ladderl pr Override Settings Std Ladder 2 Std Ladder 3 Std Ladder 4 Standard Remove Restore Original Click Import to import an analysis settings file This will be explained in more detail in Importing Analysis Settings on page 386 Click Export to export the current analysis settings file This will be explained in more detail in Exporting Analysis Settings on page 387 e Click Restore Original to restore Compass default settings Click OK to save changes and exit Compass Software for Peggy User Guide Standards Settings page 379 Click Cancel to exit without saving changes Standards Analysis Settings Groups Standards settings are saved as a group and multiple settings groups can be created Specific group settings can then be applied to individual capillaries sample names or attributes in the run data NOTES ProteinSimple recommends using the Compass default values for standards analysis settings These set tings are included in the default Standards group Analysis settings are run file specific However settings can be imported or exported for use with other run files For more information see Importing and Exporting Analysis Settings on page 386 Standards groups are displayed in the analysis settings box Analysis Settings Std Ladder premix pl 5 10 Std Ladder premix pl 4 7 Std Ladder3 premix pl 5 8
63. Print p Print Protocol Frint Template Exit The assay template will print as an image exactly as it is shown in the Template pane Importing and Exporting Protocols and Templates The assay protocol in an open assay or run file can be exported as a separate file as can the assay template annotation information This allows the same assay protocol and template information to be imported into another assay at a later time rather than having to re enter assay protocol and template information manu ally Importing an Assay Protocol NOTE Importing an assay protocol imports information into the Protocol pane only 1 Open the assay you want to import the assay protocol in to 2 Select File in the main menu and click Import Protocol 3 Select a protocol file protocol and click OK The imported information will display in the Protocol pane Compass Software for Peggy User Guide page 92 Exporting an Assay Protocol Chapter 4 Charge Assays NOTE Exporting an assay protocol exports information in the Protocol pane only 1 Open the assay you want to export the assay protocol from 2 Select File in the main menu and click Export Protocol The following window displays Organize v New folder Favorites E Desktop m3 Downloads Recent Places BB Desktop Libraries 5 Documents E My Documents di Compass Ji Assays B New Assays d Runs Documents library Assays Name _ Standard sample p
64. SS 355 GO 63 G2 G3 G4 655 G6 GF GS 659 70 1 pl Capillaries Cap Peak Name Position pl Height Area Area Width S N 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 NOTE The reported pl for immunodetected sample proteins in Compass may vary slightly trom predicted pls based on sample buffer and assay conditions Compass Software for Peggy User Guide page 266 Chapter 9 Charge Assay Data Analysis Software Menus Active in the Analysis Screen The following software menus are available File Edit View Instrument when Compass is connected to Peggy Window Help The File Edit and View menu options specific to the Analysis screen are described next File Menu The following File menu options are active Open Run Opens a run file Edit View Instrument Open Run i Add Run i Close Close All Save Save As Export Tables Export Spectra k Exit Add Run Opens and views other run files in addition to those that are already open Close Closes the run file currently being viewed Close All Closes all open run files Save Saves changes to the open run file Save As Saves changes to the open run file under a different file name Export Tables Exports the results for all capillaries in the run in txt format Export Spectra Exports the raw data traces for each capillary in the run in txt or cdf format Exit Closes Compass
65. Std Ladder4 premix pl 5 6 Standard Add Remove The Std Ladder groups shown contains the Compass default analysis settings for pl Standard Ladders used with each of the premixes Separation gradients for charge assays on Peggy Users can select and use one of these default groups make changes to groups or create new groups To view settings for a group click on the group name in the analysis settings box Compass Software for Peggy User Guide page 380 Chapter 9 Charge Assay Data Analysis Creating a New Standards Group 1 Select Edit in the main menu and click Analysis then click Standards in the options list 2 Click Add under the analysis settings box A new group will be created Analysis Settings Standards Standards 2 3 Click on the new group and enter anew name Analysis Settings Standards New Standards 4 Click in the first cell in the pl column in the Fluorescent Peaks table New Standards Fluorescent Peaks Position Registration 200 550 625 740 780 H000 K EIEIEI EE a 5 Enter the pl for the fluorescent standard Compass Software for Peggy User Guide Standards Settings New Standards Fluorescent Peaks gt pl Position Registration 4 0 200 6 550 6 4 625 T 740 73 780 H000 K EIEIEI EE a 6 Click in the first cell in the Position column New Standards Fluorescent Peaks pl Position Registration 4 Eh 6 550 6 4 625 7 740 T3 780
66. To resize columns Click the Experiment tab Roll the mouse over a column border until the sizing arrow appears then click and drag to resize Experiment pane column descriptions for the Peggy Charge default assay are as follows Sample Sample name If sample names were entered in the assay template Assay screen those names will display here Otherwise Sample default name will display NOTE Data notification icons will display in the sample column if Compass detected a potential analysis issue or data was manually modified by the user For more information see Compass Run Data Notifica tions and Warnings on page 294 Primary Primary antibody name If primary antibody names were entered in the assay template Assay screen those names will display here Otherwise Primary default name will display Cycle Run cycle number There are 12 capillaries in one cycle Compass Software for Peggy User Guide page 272 Chapter 9 Charge Assay Data Analysis NOTE Peggy can run up to eight cycles 12 capillaries per cycle in an experiment Each cycle is designated in the Experiment summary Cap Capillary number S Well on the assay plate used for sample e 1 Well on the assay plate used for primary antibody e 2 Well on the assay plate used for secondary antibody e 3 Well on the assay plate used for tertiary antibody if used Graph Pane Electropherogram Data Click the Graph tab to view data for immunodet
67. and image data Images Lets you view the chemiluminescent exposures taken during the run and view data for dif ferent exposures in the Analysis screen Peak Fit Lets you customize peak fit settings for sample data Peak Names Lets you enter custom naming settings for sample proteins associated with specific blocking reagents primary antibodies or attributes and have Compass automatically label the peaks in the run data Standards Lets you customize the molecular weight and positions Compass uses to identify ladder standards fluorescent standards and registration peaks as well as change the capillary used for the ladder Compass Software for Peggy User Guide Advanced Analysis Settings page 221 Advanced Analysis Settings The advanced analysis settings page lets you view and change analysis settings for samples standards and image data To access these settings select Edit in the main menu and click Analysis then click Advanced in the options list NOTE Settings can be modified in an assay prior to starting a run or in a run file once the run has finished executing Analysis changes made to an executing run will not be saved to the final run file Advanced Images Analysis Settings Analysis Settings Advanced Peak Fit Peak Names Standards Standards Peak Width Allowable Drift Sample Peak Fit Starting Width Ratio EA Image Advanced Median Fiter Threshoki Ratio Terraa Median Filter Threshold Limit Ada
68. are complete select File in the main menu and click Save As Select the New Assays folder Organize v New folder 4 Ft Favorites Documents library W Desktop New Assays m3 Downloads S Recent Places Arrange by Folder Name Date modified Type Simple Western assay 9 26 2011 3 25PM Compass Assay File 4 B Desktop 4 Libraries a 5 Documents s E My Documents d Compass Ji Assays m New Assays d Runs all lt 1 m File name Protein Test Save as type Assay File assay 4 Type the name for the new template assay and click Save 5 Select File in the main menu and click New Assay The new template assay will now be available in the drop down list File Edit Instrument Window Help Protein Test Simple Western Compass Software for Peggy User Guide page 88 Chapter 4 Charge Assays Viewing and Changing the Detection Exposures To view the current detection profile roll the cursor over the exposures cell in Protocol pane Cycle 1 Cycle 2 Cycle 3 t Sample gt Separation gt Immobilization Time sec 100 0 100 0 100 0 gt Primary 4b Time min 120 0 120 0 120 0 gt Secondary Ab Time min 60 0 60 0 60 0 gt Tertiary Ab Time min 10 0 10 0 10 0 4 Detection Well Row El Wash Load Time sec 2 0 Detection Profile 5 Exposures signal 30 0 sec signal 60 0 sec signal 120 0 sec signal 240 0 sec signal 480 0 sec
69. be explained in more detail in Importing Analysis Settings on page 386 Click Export to export the current analysis settings file This will be explained in more detail in Exporting Analysis Settings on page 387 e Click Restore Original to restore Compass default settings Click OK to save changes and exit Compass Software for Peggy User Guide page 368 Chapter 9 Charge Assay Data Analysis Click Cancel to exit without saving changes Peak Names Analysis Settings Groups Peak name settings are saved as a group and multiple settings groups can be created Specific group set tings can then be applied to individual capillaries blocking reagent names or primary antibody names and attributes in the run data NOTE Analysis settings are run file specitic However settings can be imported or exported for use with other run files For more information see Importing and Exporting Analysis Settings on page 386 Peak name groups are displayed in the analysis settings box Analysis Settings Peak Names 1 Add Remove The Peak Names group shown is a Compass template group Users can make changes to this group and cre ate new groups To view settings for a group click on the group name in the analysis settings box Creating a Peak Names Group 1 Select Edit in the main menu and click Analysis then click Peak Names in the options list 2 Click on the Peak Names 1 template group in the analysis setti
70. changes and exit Click Cancel to exit without saving changes Range Settings Minimum The molecular weight value in kDa below which peaks will not be identified This value will also be used as the default lower MW range for the data displayed in the electropherogram and virtual blot The default value is 1 kDa Maximum The molecular weight value in kDa above which peaks will not be identified This value will also be used as the default upper MW range for the data displayed in the electropherogram and virtual blot The default value is 250 kDa Baseline Settings Threshold The variance or roughness in a baseline data segment below which a point will be called part of the baseline The default value is 1 0 Window How long baseline data segments are expected to be in pixels Shorter segments will allow the baseline to follow plateau sections of the signal The default value is 15 Stiffness The amount the baseline is allowed to vary from a straight line Settings between 0 1 and 1 0 make the baseline fit closer to a straight line Settings from 1 0 to 10 0 will make the baseline fit fol low the data more closely The default value is 1 0 Peak Find Settings Threshold The minimum signal to noise ratio required for a peak to be identified A setting of 1 0 will detect many peaks a setting of 10 0 will detect fewer peaks The default value is 10 0 Width The approximate peak width at full width half max in pixels use
71. concentrations as they are also used for capillary registration In the following example the pl standards shown are those for pl Stan dard Ladder 3 P N 040 646 DemoData_Charge_HeLa_ERK12 Compass Lo File Edit View Instrument Window Help ot e e Assay CY Run Summary E Experiment 6 fe Graph E Image Lane T Br N m I 5 Sample Primary Cycle Standards HH High phos ERK1 2 1 340 a sa Low phosp ERK1 2 1 20 ki m High phos ERK1 2 1 300 Low phosp ERK1 2 1 i 280 High phos ERK1 2 1 260 Low phosp ERKI 2 1 a High phospho He pie 8 High phos ERKI 2 1 Low phosp ERKI 2 1 ea SEa High phos ERKI 2 1 2 200 190 Low phosp ERK1 2 1 High phos ERKI 2 1 5 160 Low phosp ERK1 2 1 i 140 120 100 80 2 Std 6 0 40 ee Std 7 0 20 y 0 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 80 850 900 950 1 000 Position E paaka Capillaries e Primary Peak Position Height ERK1 2 7 271 10 9 ERK1 2 8 314 202 4 ERK1 2 9 671 29 6 ERK1 2 10 745 26 0 ERK1 2 11 874 ERK1 2 12 886 ERK1 2 13 918 If standards are not identified correctly they can be manually corrected as follows Ifan incorrect peak is identified as a standard Right click the peak in the electropherogram or peaks table and select Not a Standard Compass should correctly reassign the remaining peaks as standards and update the peaks table Compass Software for Pe
72. data displayed in the Analysis screen is compiled from all exposures taken during the run and utilized to calculate the chemiluminescent signal output at time zero of the chemiluminescent reaction This calculation represents the kinetics of the chemiluminescent reac tion and helps to eliminate signal burn out that may occur with stronger signals and longer exposure times Exposure Sample data displayed in the Analysis screen is for this specific exposure only To see the number of exposures and exposure times used for the run data click the arrow in the drop down list next to All Compass Software for Peggy User Guide page 230 Cycle z Luminescence Exposure 30 seconds Exposure 2 60 seconds Exposure3 120 seconds Exposure4 240 seconds Exposure 480 seconds ExposureG 960 seconds Chapter 8 Size Assay Data Analysis NOTE The number of exposures taken and exposure times shown were specified in the assay protocol Assay screen prior to executing the run and cannot be modified Changing the Sample Data Exposure Displayed The exposure used for the sample data displayed in the Analysis screen can be changed To do this 1 Select Edit in the main menu and click Analysis then click Images in the options list 2 Click the arrow in the drop down list next to All and select an exposure setting Compass Software for Peggy User Guide Cycle All Luminescence Multi Image Analysis Exposure 30 se
73. eee er ee eee eee 293 Step 2 Checking Fluorescent Sizing I TA AE EEIE EE A MOE AE EE 296 Step 3 Checking Capillary Registrations 299 Step 4 Checking Samne Sse ne iT 301 Step 5 Assigning Peak Names Optional 303 GOURS S E e EAE EE AEEA 304 UO HOUDS 5 3 nd xg Moma EE 304 VEW STOUSUCS ee wate n iain cet 305 Hiding or Removing Capillaries in Group ANOS sass tiiek EA ann thon 308 Copying Data Views and Results Tables 310 CODVING DOT W adar S10 COV WAG RESUS MOINES araa arr tv luild Sob adetate tastes 310 Saving the Graph View as an Image File 310 PPOR OF NOS csisccii Rs ees a OAT Dae page vi EXDOMING TCS UIE GOES usta TEE S 311 Exporting Raw Sample Electropherogram B Eo TEPEE EI EIE E E A EOT oye Changing Sample Protein Identification 312 Adding or Removing Sample Datd 312 FUICIOG SOA DIC ONG sii ieee toa abacus aac 314 Changing Peak Names for Sample Data 315 Displaying Sample Data for Named Peaks OY EEE EE E E E EE ETT O E 317 Changing the Virtual Blot VieW tacsis eiriaa ias 319 Ading RECONTO Cis ia tiudtte s oa a rare a 319 Inverting the Virtual Blot 0 c cece 320 SEICCUIG LODE LOO CIS EEIE 320 Viewing the Uncorrected Sample Baseline 32 Overlaying Standards Data on Sample Lanes 322 Moving Lanes in the Virtual Blot View 324 Changing the Electropherogram View 525 Autoscaling the Electropherogram
74. either the graph or lane panes but manual corrections must be done in the graph pane Graph Pane a Click Single View in the View bar b Click on the row in the experiment pane that contains the sample you wish to check then click the Graph tab Compass Software for Peggy User Guide page 302 Chapter 9 Charge Assay Data Analysis DemoData_Charge_HeLa_ERK12 Compass Window Help ta File Edit View Instrument Seas ESL Assay o Run Summary 4 ie E Experiment Primary ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 Sample Cycle High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos Low phosp High phos ee Low phosp Chemiluminescence oSS8BSR2S8 85888 88 82 8 Sample aph gt 5 Image E Lane S N HH Capillaries ppErk1 pErk1 Erk1 ppERK2 Ui Gi IN ria EL ke pl Samples ry te th LeO a 6 1 6 3 64 65 High phospho HeLa in 5 8 Cii ES T GJ 68 E3 ered Primary Cap Peak Name Position Height Area Area nat 62 4 oe 1538 7 If sample peaks are not identified correctly they can be manually corrected as follows Ifan incorrect peak is identified as a sample peak Right click the peak in the electrophero gram or peaks table and select Remove peak Compass
75. file data click the arrow in the drop down list next to Default then click the new group from the list Analysis settings in the new group will then be applied to the run data Analysis Settings Advanced STAT analysis Default Advanced Override 6 Click OK to save changes Changing the Default Analysis Group 1 Select Edit in the main menu and click Analysis then click Advanced in the options list 2 Click the arrow in the drop down list next to Default then click a new default group from the list Analysis Settings Advanced STAT analysis Default Override Compass Software for Peggy User Guide Advanced Analysis Settings page 225 3 Click OK to save changes Analysis settings in the group selected will be applied to the run data Modifying an Analysis Group 1 Select Edit in the main menu and click Analysis then click Advanced in the options list 2 Click on the group in the analysis settings box you want to modify Analysis Settings Advanced 3 Modify standards sample or image parameters as needed 4 Click OK to save changes The new analysis settings will be applied to the run data Deleting an Analysis Group 1 Select Edit in the main menu and click Analysis then click Advanced in the options list 2 Click on the group in the analysis settings box you want to delete and click Remove Analysis Settings Advanced STAT analysis 3 Click OK to save changes Ap
76. file includes a history of signed changes to the file Compass can be run with or without Access Control enabled When Access Control is disabled no user log in is required and files are not encrypted or signed The instrument history and file history are still maintained but the entries are not signed Compass Software for Peggy User Guide Enabling Access Control page 403 Enabling Access Control Access Control is enabled in Preferences Select Edit in the main menu click Preferences then select Access Control type filter text Access Control SESS ala Enable Analysis Graph Server 10 1 2 18 Restore Defaults Apply To enable Access Control 1 Check the Enable box 2 Enter the IP address of the Authorization server Use format X X X X or LocalHost if installing the server on the local machine NOTE Always use the default port setting of 8000 this should not be changed 3 Close Compass The next time Compass is launched a user log in will be required NOTE Access Control can only be disabled by logging into Compass and deselecting the Enable box in the Access Control page of Preferences Compass Software for Peggy User Guide page 404 Chapter 11 Compass Access Control and 21 CFR Part 11 Compliance Logging In to Compass With Access Control enabled all users must log in to Compass whenever the software is launched Enter your user name and password previously setup by your
77. files Save Saves changes to the open run file Save As Saves changes to the open run file under a different file name Export Tables Exports the results for all capillaries in the run in txt format Export Spectra Exports the raw data traces for each capillary in the run in txt or cdf format Exit Closes Compass Compass Software for Peggy User Guide Analysis Screen Overview page 135 Edit Menu The following Edit menu options are active View Instrument Windo Cut Copy Ctrl C Paste Ctrl V Analysis Preferences Copy Lets you copy data shown in the graph lane peaks or capillaries panes See Copying Data Views and Results Tables on page 181 for more information Analysis Displays the analysis settings used to analyze the run data and lets you change them as needed See Compass Analysis Settings Overview on page 219 for more information e Preferences Lets you set and save custom preferences for data export plot colors in the graph and Peggy s Twitter settings See Chapter 10 Setting Custom User Preferences for more information View Menu The following View menu options are active Instrument Window Single View Multiple View Standards Registration Samples Filter View Region Show Hidden Single View Displays data in a per capillary single view format e Multiple View Displays data in a per 12 capillary multiple view format Standards
78. fluorescent standards only Standards are highlighted in both the peaks and capillaries tables and marked with an S For information on checking and identifying standards peaks see Step 2 Checking Fluorescent Sizing Standards on page 164 To view registration data Click Show Registrations in the View bar or select View in the main menu and click Registration Sr wit 51 File Edit View Instrument Window Help i7 Assay cry Run Summary Sample biotin hela hela hela hela hela hela hela hela hela hela biotin hela hela hela hela hela hela hela hela hela hela hela biotin hela hela hela hela hela hela hela hela hela hela hela biotin hela hela hela ES W m pars Primary Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki a Graph E Image Lane Registrations Hm Fluorescence 500 Position E Peaks H Capillaries Blocki C2 12 Blocki C2 12 Blocki C2 12 Blocki C2 12 Blocki C2 12 1 Primary Cap Peak Position
79. in the electrophero gram or peaks table and select Remove peak Compass will no longer identify it as a sample peak in the electrooherogram and the peak data will be removed in the results tables To set an unidentified peak as a sample peak Right click the peak in the electropherogram or peaks table and select Add Peak Compass will calculate and display the results for the peak in the results tables and identify the peak in the electropherogram NOTE To remove sample peak assignments that were made manually and go back to the original view of the data right click in the electropherogram and click Clear All Compass Software for Peggy User Guide page 174 Chapter 8 Size Assay Data Analysis c Repeat the previous steps for the remaining sample rows in the experiment pane to make sure all sample proteins are identified correctly Lane Pane a Click either Single View or Multiple View in the View bar b Click on the row in the experiment pane that contains the sample you wish to check then click the Lane tab To view sample protein band labels roll the cursor over the individual bands If sample bands are not identified correctly they must be corrected in the graph pane as described in the pre vious section secs File Edit View Instrument Window Help tam t gt Brey Consonnes z A MEE Graph BA Image E Lane _ Ha Ss Sample Primary biotin Blocki hela Blocki hela Blocki hela Block
80. is aspirated Capillaries are then transferred to an incubator tray When incubation is complete Wash Buffer is aspirated Secondary Antibody 2 Step Capillaries are moved to the assay plate in the sample tray and secondary HRP conjugate and Streptavidin HRP is aspirated Capillaries are then transferred to an incubator tray When incubation is complete Wash Buffer is aspirated Detect Step Capillaries are moved to the assay plate in the sample tray and Luminol Per oxide solution is aspirated Capillaries are then transferred to the separation tray where the emitted chemiluminescent light is detected with the CCD camera Compass Software for Peggy User Guide Viewing File and Run Status Information page 113 Description Results Step Results are available in the Analysis screen Hold Step This step is used for overlapping or overlapping with hold schedule options A cycle that has completed a specific step will go into a hold step while the remaining cycles execute the same step in parallel For example once cycle1 completes the separate step it may go into a hold step while cycle 2 executes the separate step This execute and hold pattern will continue until all cycles have completed the separate step When a run is in progress icons for steps that have not executed will be grey inactive In the following example the Secondary Antibody 2 step is executing and the Detect and Results steps have not started sampl
81. kDe FPP LFF PIP PPIF FEF FPPEPPPEPPEPEPPST EE Peaks EE Capillaries Sample Primary Name Position MW kDa Height Area Width S N biotin lad Blocki Ldr 12 413 522 8 23 1 87 1 biotin lad Blocki gt Ldr 40 629 699 7 220 1212 biotin lad Blocki Ldr 66 739 682 5 183 1474 biotin lad Blocki Ldr 90 776 1598 4 17 2 3548 biotin lad Blocki Ldr 116 8 1039 1 biotin lad Blocki 88 16 hela Blocki cit 12 116 21661 196 2001 ca Ldr 180 7 180 609 7 559 255 84 0 a2 3 955 250 2231 3604 152 913 hela Blocki hela Blocki hels Rlacbi ai m hela E gt FP FPF RPWWWhWwWWHswsnswswswwnwwnwnwnnnnnn nny nN NNN N e FP PP PP RP PP eR gt Screen Panes Assay Run Summary and Analysis screens each contain multiple screen panes that allow users to view the individual components of a run assay or data file Each pane has a labeled tab and a unique icon The panes specific to each screen will be described in the individual screen sections Compass Software for Peggy User Guide page 6 Chapter 1 Getting Started with Compass The active pane in a screen is blue To view a pane click in the pane or on its tab The example below shows panes in the Analysis screen and the Lane pane is active Title Bar The title bar displays the run file name and allows the main Compass window to be minimized maximized or closed spe we ba Co a Main Me
82. of multiple open run files In the experiment pane click on one of the sample rows in the file Next click File from the main menu and click Close e To close all open run files Select File from the main menu and click Close All Compass Software for Peggy User Guide Compass Analysis Settings Overview page 219 Compass Analysis Settings Overview Compass has a variety of analysis features and settings that users can modify as needed to enhance run data To access these settings select Edit in the main menu and click Analysis If more than one run file is open select the run file you want to view analysis settings for from the list Edit View Instrument Window Help Cut Copy Ctrl C Paste Ctrl V Analysis Simple Western ERK Demo Preferences 2011 07 13 16 55 20 _7 12 2011 The following screen will display Advanced Images Analysis Settings Analysis Settings Advanced Peak Fit Peak Hames Standards Standards Peak Width Allowable Drift Sample Peak Fit Starting Width Ratio Image Default Advanced Merdan Fiter Theshoki Ratio ank Median Filter Threshold Limit Restore Original Compass Software for Peggy User Guide page 220 Chapter 8 Size Assay Data Analysis To move between analysis pages in this window click on an option in the list on the left The following anal ysis settings can be user customized in Compass Advanced Lets you customize analysis settings for samples standards
83. of these runs or click Browse to open the Runs folder and select a different file When a run is added its data will append to the open run file and will display as a second set of up to 96 capillaries in all screen panes The second run file name will also appear in the Compass title bar Compass Software for Peggy User Guide page 138 File Edit View Instrument Window Help Chapter 8 Size Assay Data Analysis eet e 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 Name Ldr12 Ldr 40 Ldr 66 Ldr 90 Ldr 116 Ldr 180 5 Repeat the last two steps to open additional runs Compass Software for Peggy User Guide Position 402 624 730 766 801 MW kDa 116 1459 7 1008 0 2120 8 3749 4 2032 5 Biotin La Blocki Gia 877 180 1046 4 27077 24 _ _ How Run Data is Displayed in the Analysis Screen page 139 How Run Data is Displayed in the Analysis Screen Data in the run file is cCompartmentalized for easy review in one of six panes in the Analysis screen Experiment Pane Assay and Capillary Information The experiment pane displays assay and capillary information for each of the 96 capillaries in the run A maxi mized view of the experiment pane is shown below E Experiment Sample Primary Cycle Cap 5 1 2 F Biotinylated Ladder Blocking 1 1 c1 D1 El Fl wf K562 anti EREL 2 1 2 C2 D2 E2 F
84. opened will display Select one of these runs or click Browse to open the Runs folder and select a different file Opening Multiple Run Files NOTE If you need to open a run file when another run is executing launch another instance of Compass first then open the file 1 To open the first run file select File in the main menu and click Open Run Compass Software for Peggy User Guide Opening Run Files Edit Open Run Sally NFKB pathway test 2 Add Run Sally NFkB pathway test 1 Sally HeLa IKb test 2012 03 05_11 51 19_HelaControlERKassay 2012 03 05_14 45 07_2012Mar05Multi Western_IkB 2012 03 14 _HeLa_ERK STAT3 PLCg Day5_PLOO3 Copy Sally HeLa Day 3 Run Sally HeLa TNF treatment Sally MCF Multi target screen 2012 03 12_MCFisc_PBK p110b cs3011_filtAD_PLOO3 Instrument Window Close Close All Instrument Sally Sall started Yved 8 20 F Completed Thu 2 57 F page 137 2 Alistofthe last 10 runs opened will display Select one of these runs or click Browse to open the Runs folder and select a different file 3 To open another run file select File in the main menu and click Add Run File Edit View Instrument Window Help Open Run i Add Run 2011 08 31 _16 38 23_test 3 ab run 2011 09 01_16 41 02 Close Close All Save iambic Save As Export Tables Export Spectra Exit 4 Alist of the last 10 runs opened will display Select one
85. pane can be copied and d into other documents as can the graphic image of the annotations in the Template pane Copying an Assay Protocol 1 Click on the Protocol tab 2 Select Edit in the main menu and click Copy 3 Open a document Word Excel etc Right click in the document and select All assay protocol steps and parameters will be copied into the document as a list in the same format shown in the Protocol pane Compass Software for Peggy User Guide Printing Protocols and Templates page 47 Copying an Assay Template 1 Click on the Template tab 2 Select Edit in the main menu and click Copy 3 Open a document Word Excel etc Right click in the document and select The assay template will be copied into the document as an image exactly as it is shown in the Template pane Printing Protocols and Templates The information in the Protocol pane can be printed as can a graphic image of the information in the Tem plate pane Printing an Assay Protocol 1 Click on the Protocol tab 2 Select File in the main menu click Print and then click Print Protocol Edit Instrument Window Help New Assay j Open Assay li amme Pro Save Save As gt m Import Frotocol Sa Import Template Se Export Protocol Ir Export Template 7 Se Print Print Protocol Exit Frint Template All assay protocol steps and parameters will be printed as a list in the same format shown in the Protocol pane Printing
86. respectively Compass Software for Peggy User Guide Changing the Electropherogram View page 345 Standards Checking this box aligns the pl of the raw standards data to the sample data and over lays both electropherograms in the graph pane ke Gra ph g Image Lane EF B ce oa i ppErk1 pErk1 Erk1 pERK ERK ppERK2 Hi shospho HeLa 1 1 ss SSSSSeES Chemilumines cence gt o 8 Pl a vel aes A i Ju a0 51 52 53 54 55 56 57 58 59 EO El G2 E3 E4 GS EG ET E8 E9 7O pl Adding and Removing Baseline Points Points in the baseline can be added or removed as needed To do this 1 Click the Graph Options button in the graph pane toolbar and check Baseline Fit This will display baseline points for the raw sample data 2 Use the mouse to draw a box around the area you want to correct This will Zoom in on the area Compass Software for Peggy User Guide page 346 Chapter 9 Charge Assay Data Analysis 3 Right click a baseline point and click Add Baseline Point or Remove Baseline Point N 2 Image Lane t eE res m ppErk1 pErk1 ppERK Low phaspho HeLa C1 4 Chemilumines cence Zoom Out Add Peak Add Baseline Point Remove Baseline Point Clear All 950 200 20 200 790 700 650 600 10 500 450 400 ao Copy Ctrl C NOTE To clear the manual addition or removal of baseline points and go back to the original view of the data right click in the electropherogram and cl
87. sent to the instrument that were signed by the user who sent the command If you want to copy the Command Log at any time right click in the table and select Copy then paste into another document Date 05 01 2013 4 07 PM 05 02 2013 1 57 PM 05 02 2013 3 46 PM 05 03 2013 3 00 PM 05 03 2013 3 00 PM 05 03 2013 3 03 PM 05 03 2013 3 03 PM 05 03 2013 3 03 PM 05 03 2013 3 03 PM 05 03 2013 3 06 PM 05 07 2013 3 31 PM 05 08 2013 4 46 PM 05 09 2013 4 25 PM 05 10 2013 3 46 PM Chapter 11 Compass Access Control and 21 CFR Part 11 Compliance Message Started run Run 2013 05 01_15 50 32_models_R Started Self Test Started run Run 2013 05 02_15 29 13_models_ Started run Run 2013 05 03_14 43 31_Screening Reset after error Started run Run 2013 05 03_14 45 37 Screening Stopped run Stopped run Stopped run Started run Run 2013 05 03_14 49 52 Screening Started run Run 2013 05 07_15 14 15_DExinpri Started run Run 2013 05 08_16 29 35 TrisT22_2 Started run Run 2013 05 09_16 08 00_BGAR_Rb Started run Run 2013 05 10_15 28 43 E 7assay_P 05 01 2013 4 07 PM Started run Run 2013 05 01_15 50 32_models_Rb3step_Peggysize_TrisTricine22 Assay Compass Software for Peggy User Guide uyen Cancel Run File History page 409 Run File History Select the Run Summary screen tab and then the History tab to see the file History To copy the file History right click in the table and select Copy
88. settings box you want to apply to specific run data Analysis Settings 3 Application of peak names groups to specific run data is done in the override box Click Add under the override box A default override data set will be created Apply Settings Settings ERKI Add Remove 4 Click the cell in the Apply To column then click the down arrow Compass Software for Peggy User Guide Peak Names Settings page 375 Apply Settings Custom Settings 5 Select an option from the drop down list This will apply the settings group selected to specific run data as follows e All When this option is selected peak names group settings will be applied to all capillaries in the run file Primary antibody names All primary antibody names entered in the assay template Assay screen will display in the drop down list otherwise the default name of Primary will be shown Select aname to apply group settings to all capillaries that use the primary antibody name in the run file Attributes All primary antibody attributes entered in the assay template Assay screen will dis play in the drop down list Select an attribute to apply group settings to all capillaries that use this attribute in the run file Cycle 1 8 When this option is selected group settings will be applied to all capillaries in the cycle Custom settings Lets you choose specific capillaries to apply the group settings to When this option is
89. system properties which include Name Location Type Serial number Instrument software version firmware Network name and add ress Date and time of the instrument clock Compass Software for Peggy User Guide Viewing Log Files page 125 Peggy SW1002 Properties Name Peggy SW1002 Location Type Peggy Network Name sw1002 local Serial Number SW1002 Network Address 10 1 2 158 Instrument Software 2 016919 Instrument Date and Time 2012 10 18 12 45 29 07 00 Set to PC time View Error Log View Test Log Tochange system name or location click in the name or location boxes and enter the new infor mation To sync the instrument clock with the computer click Set to PC time Viewing Log Files Error Logs 1 Select Instrument in the main menu and click Properties to display system properties 2 Click View Error Log A list of system logs will display Compass Software for Peggy User Guide page 126 D PLOOOS Logs e g Name embedded log temperature log temperature log 2012 10 29 lamp log error log temperature log 2012 10 28 temperature log 2012 10 27 temperature log 2012 10 26 temperature log 2012 10 25 stdout log temperature log 2012 10 21 temperature log 2012 10 20 embedded log 1 embedded log 2 embedded log 3 embedded log 4 Date 2012 10 30 13 59 2012 10 30 13 58 2012 10 30 09 39 2012 10 30 03 28 2012 10 29 17 06 2012 10 29 09 39 2012 10 28 09 38 2012 10 27 09 37
90. to 96 capillaries per experimental run Experiment Lists the assay protocol steps and assay template information e Graph Displays electropherogram data for immunodetected sample proteins fluorescent standards or capillary registrations e Image Displays a 12 capillary image of the separated immunodetected sample proteins fluores cent standards or capillary registrations e Lane Displays data for immunodetected sample proteins fluorescent standards or capillary registra tions as bands in individual lanes This virtual blot like image is similar to traditional blot results e Peaks Lists the tabulated results for immunodetected sample proteins and information for identi fied fluorescent standards and capillary registrations Capillaries Displays a list of the immunodetected sample proteins Compass named automatically using the user defined peak name analysis parameters Compass Software for Peggy User Guide Analysis Screen Overview page 265 DemoData_Charge_Hela_ERK12 Compass File Edit View Instrument Window Help Pest ElMu D Primary ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 pErk1 Erk1 E Chemilumines cence oS8SBSRSR8SRS8 8888 8 50 5 52 33 34 55 S56 SY
91. with this option as proteins are locked to the capillary wall and therefore more stable than samples on the assay plate This option also results in the most signif icant reduction in overall run time NOTE ProteinSimple recommends using the overlapping with hold option For more information on when to use other schedule options please contact ProteinSimple Technical Support Step 6 Add Assay Plate Annotations Optional Custom reagent names notes and annotations can be entered for individual rows and wells on the assay plate This information will be stored with assay and run data During post run analysis this information will be used to apply custom analysis settings to specific sample names blocking reagent names primary anti body names or sample and primary antibody attributes This will be explained in more detail in Compass Analysis Settings Overview on page 219 NOTE Template pane information can also be added or updated after a run is complete Compass Software for Peggy User Guide Creating a New Assay page 37 To enter annotations 1 Click on the Template tab The default annotations for reagent rows and individual wells on the assay plate will display Stacking Matrix Compass Software for Peggy User Guide page 38 Chapter 2 Size Assays 2 Change or add row and well annotations as needed To do this a To enter annotations for a specific well Right click the well and select Edit
92. 0 Cancel 6 Ifyou need to change the peak fit group used for a data set click the cell in the Settings column and click the down arrow Select a group from the drop down list Override Apply To Settings Hi phospho HeLa STAT peak fit STAT peak fit 7 Repeat the previous steps to apply other groups to specific run data 8 To remove a data set click on its cell in the Apply To column then click Remove 9 Click OK to save changes Compass Software for Peggy User Guide Peak Names Settings page 367 Peak Names Settings The peak names analysis settings page lets you enter custom naming settings for sample proteins Compass can automatically label sample peaks in the run data associated with specific blocking reagent names primary antibody names or attributes To access these settings select Edit in the main menu and click Analysis then click Peak Names in the options list NOTE Settings can be modified in an assay prior to starting a run or in a run file once the run has finished executing Analysis changes made to an executing run will not be saved to the final run file oS Analysis DemoData_Charge_HeLa_ERK12 cbz Peak Names Advanced Images Analysis Settings Analysis Settings ERKL Peak Fit ERKI Pak Hame mm pl Color Range Show Standards 55 0 1 E 0 0 5 E Apply Settings Apply To All All Remove Remove Restore Original Click Import to import an analysis settings file This will
93. 0 0 120 0 120 0 Well Row Sal asa a ae ae Washes 2 2 2 2 2 2 2 2 Wash Soak Time sec 150 0 150 0 150 0 150 0 150 0 150 0 150 0 150 0 Secondary Ab Time min 60 0 60 0 60 0 60 0 60 0 60 0 60 0 60 0 Detection 4 f needed change the primary incubation reagent row location To do this click the cell in the value col umn next to Well Row and select a different row on the assay plate Protocol Ts No Add Remove Cyclel Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle 8 Separation Matrix Stacking Matrix Sample Separation Time min 40 0 40 0 40 0 40 0 40 0 40 0 40 0 40 0 Separation Voltage volts 250 250 250 250 250 250 250 250 Matrix Removal Blocking Time min 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 Primary Ab Time min 120 0 120 0 120 0 120 0 120 0 120 0 120 0 120 0 Well Row Washes Wash Soak Time sec Secondary Ab Time min Detection cl 60 0 a a 2 Fd 2 2 2 2 2 2 150 0 60 0 150 0 60 0 150 0 60 0 150 0 60 0 150 0 60 0 150 0 60 0 150 0 60 0 NOTE Only rows designated as primary antibody in the Layout tab can be selected in the Well Row drop down menu If needed change the secondary incubation time To do this click the cell in the value column next to Secondary Ab Time min and enter a new value in minutes Compass Software for Peggy User Guide Creating a New Assay page 33 Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle am
94. 00 600 500 400 300 200 4 o 8 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 pl Baseline and Grid Options You can view the calculated baseline fit peak integration and show grid lines with these options E al Fitted Peaks E J amp Baseline Fit E Grid Lines e Fitted peaks Checking this box will display how the peaks were fit by the software NOTE This option is only available for sample data Compass Software for Peggy User Guide Changing the Electropherogram View page 335 fr Graph a Image Lane T BF B ana Al perki Erki ppERK2 Chemilumines cence 300 200 700 600 500 400 300 200 50 51 52 53 54 55 56 57 58 59 60 61 62 63 G4 65 66 67 68 69 70 pl Baseline Fit Checking this box will display the calculated baseline for the peaks Baseline points will also display for regions of the electrooherogram considered to be at baseline Compass Software for Peggy User Guide page 336 Chapter 9 Charge Assay Data Analysis NOTE This option is only available for sample data Hi phospho HeLa C13 1 u TI T TI wo a E 3 E D a tbh bt hh 630 635 640 645 650 655 660 66 67 675 680 68 69 695 7 00 pl Compass Software for Peggy User Guide Changing the Electropherogram View page 337 Grid Lines Checking this box will display grid lines in the graph area kn Graph BS Image Lane t Be B e Samples 16004 ppErkt pErk1 Erk4 p
95. 1 1 Support for new instrument Simon Viewing the Software Log Select Help and click Compass Log to view the software log file Compass Version Information Select Help and click About Compass to view the software version and build number information Compass Version 2 3 1 Build ID 1008 Compass is the control and data analysis application for NanoPro Simon Sally and Peggy instruments Copyright 2009 2012 ProteinSimple All Rights Reserved Installation Details Compass Software for Peggy User Guide page 16 Chapter 1 Getting Started with Compass Directory and File Information The main Compass directory is located in the Program Files folder and also contains a PDF file of the Peggy User Guide gt Computer gt Local Disk C Program Files x86 Compass gt File Edit View Tools Help Organize v Include in library Share with Burn Compatibility files New folder gt jy Intel z Name Date modified Type Size E a a configuration 10 11 201212 06PM_ File folder ne vesgenecac Examples 10 11 2012 12 06 PM File folder A HE features 10 11 2012 12 06 PM File folder q jre 10 11 201212 06 PM File folder ides p2 10 11 201212 06 PM File folder plugins 10 11 2012 12 06 PM File folder Soci rae ma readme 10 11 201212 06 PM File folder bid Cisco templates 10 11 201212 06 PM _File folder De Citrix eclipseproduct 7 29 2010 10 36 AM ECLIPSEPRODUCT 1KB bigs Common Files artifactsxml 10
96. 1 Select Edit in the main menu and click Analysis then click Peak Names in the options list 2 Click on the group in the analysis settings box you want to modify Analysis Settings 3 Change the information in the analysis settings peak table as described in Creating a Peak Names Group on page 239 4 Click OK to save changes Deleting a Peak Names Group 1 Select Edit in the main menu and click Analysis then click Peak Names in the options list 2 Click on the group in the analysis settings box you want to delete and click Remove Compass Software for Peggy User Guide Peak Names Settings page 245 Analysis Settings 3 Click OK to save changes Applying Peak Names Groups to Run Data 1 Select Edit in the main menu and click Analysis then click Peak Names in the options list 2 Click on the group in the analysis settings box you want to apply to specific run data Analysis Settings 3 Application of peak names groups to specific run data is done in the override box Click Add under the override box A default override data set will be created Apply Settings Apply To Settings All ERKL 2 Add Remove 4 Click the cell in the Apply To column then click the down arrow Compass Software for Peggy User Guide page 246 Chapter 8 Size Assay Data Analysis Apply Settings Apply To Settings ERKL 2 Custom Settings 5 Select an option from the drop down list This will apply the
97. 1 Select Instrument and click Open Trays amp Open Trays Main Trays Other Trays 2 Click Separation to open the separation tray 3 Add 800 uL of deionized water to the troughs in the separation tray and soak for 20 minutes 4 Remove the water by either aspirating with a pipette or with the vacuum wand located on the inside of Peggy s left door 5 Repeat the steps above until the Running Buffer or residues are completely removed 6 To complete the cleaning process select Instrument and click Cleanup Compass Software for Peggy User Guide page 124 Self Test Peggy can perform a series of self tests to check for proper instrument performance To start the test select Instrument and click Self Test The test takes approximately two minutes Chapter 7 Controlling Peggy Self Test Compass File Edit Instrument Window Help Testing Stop p Sat 4 53 PM Run Self Test TY Status Run Self Test Assay Self Test Instrument Started Peggy Peggy SW1001 SW1001 Sat 4 53 PM Jun 9 2012 PDT Sat 4 55 PM aey ty os Separation t N Plot im NOTE ProteinSimple recommends performing the self test prior to starting a run To view the test log at completion of the test select Instrument click Properties and click View Test Log See Self Test Logs on page 128 for more information Viewing and Changing System Properties Select Instrument and click Properties to display
98. 1 52 53 54 55 55 S57 58 55 G60 GI G2 63 64 G5 65 GY 5B GS 70 71 pl Sample Cap Peak Name Position Height Area Width 5 Repeat the last two steps to open additional runs 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Compass Software for Peggy User Guide How Run Data is Displayed in the Analysis Screen page 271 How Run Data is Displayed in the Analysis Screen Data in the run file is Compartmentalized for easy review in one of six panes in the Analysis screen Experiment Pane Assay and Capillary Information The experiment pane displays assay and capillary information for each of the 96 capillaries in the run A maxi mized view of the experiment pane is shown below E Experiment a Sample Primary Cycle Cap 5 1 2 3 High phospho HeLa in 5 8 ERK1 2 1 1 Al fT AL Low phospho HeLa in 5 8 ERK1 2 1 2 A P A High phospho HeLa in 5 8 ERK1 2 1 3 A B An Low phospho HeLa in 5 8 ERKI 2 1 4 4 BH An High phospho HeLa in 5 8 ERKI 2 1 5 45 D Aw Low phospho HeLa in 5 8 ERK1 2 1 6 46 Ib An High phospho HeLa in 5 8 ERK1 2 1 T A TF Aw Low phospho HeLa in 5 8 ERK1 2 1 amp 46 amp An High phospho HeLa in 5 8 ERK1 2 1 g 49 BD AL Low phospho HeLa in 5 8 ERK1 2 1 10 A D0 A High phospho HeLa in 5 8 ERKI 2 1 li A M A Low phospho HeLa in 5 8 ERK1 2 1 1 lt A D2 A To view all columns Click the Experiment tab then use the scroll bar or click Maximize in the upper right corner
99. 100 ak 0 J Fi si s2 s3 a an ae ar eS bO EI ca ca ed es eE E7 cS bg M pl Users can customize the colors used for the overlay plot display For more information see Selecting Custom Plot Colors for Graph Overlay on page 392 Compass Software for Peggy User Guide page 200 Chapter 8 Size Assay Data Analysis Zooming To zoom in on a specific area of the electropherogram hold the mouse button down and draw a box around the area with the mouse Sam ples D Lend Ti E E D l D MW kDa Compass Software for Peggy User Guide Changing the Electropherogram View page 201 To return to default scaling right click in the electrooherogram and click Zoom Out 588 Zoom Out Add Peak Add Baseline Point Remove Baseline Point Clear All TI a o wo a E 3 T E O Copy Ctrl C sgeggags ak MW kDa Customizing the Data Display Electropherogram peak labels plot labels and display options can be customized by the user To do this select the Graph Options button Compass Software for Peggy User Guide page 202 EE Graph gt 4 Image Lane l oS SSS S828 85 Chemilumines cence Peak Labels Samples ERK2 F 40 MW kDa RK 1 Chapter 8 Size Assay Data Analysis TT Matching Peak Names Peak Names Peak Values E al Fitted Peaks E J Baseline Fit Grid Lines Plot Label Sample E Attribute El
100. 11 Compliance Use the Groups page to change the permissions in a group or create new groups Site administration Prote x W 7 gt O 10 1 2 18 8000 admin Welcome admin Change password Log out Site administration Recent Actions Add Change My Actions Add Change steveg User WF steveg User TypeScientist User TypeScientist User TypeScientist User TypeReviewer User P TypeRewiewer User fay To change permissions for a group click Change then select a group T Select group to change F x E gt O 10 1 2 18 8000 admin auth group Welcome admin Change password Log out Home gt Auth gt Groups Select group to change qt O O O O O e C Group C Operator Reviewer Scientist 3 groups Move individual group permissions in or out of the Available Permissions and Chosen Permissions boxes by selecting a permission in either box Click the left or right arrow button to move the permission into the other box Compass Software for Peggy User Guide Authorization Server page 417 Change group Proteinsir x W 1 E 4 10 1 2 18 8000 admin auth group 3 Reload this page Welcome admin Change password Log out Home gt Auth gt Groups gt Scientist Change group Name Scientist Hold down Control or Command on a Mac to select more than one Permissions Available permissions sio q Filter llow analysis editing llow copy export of da
101. 144 Chapter 8 Size Assay Data Analysis Name Peak name Displays peaks that Compass named automatically using the user defined peak name analysis parameters Cells will be blank if Compass was not able to name the peak or if naming parameters were not entered Position Displays the pixel position of a peak in the image MW kDa Displays the calculated molecular weight in kDa for the peak shown for sample data only Height Displays the calculated peak height Area Displays the calculated peak area shown for sample data only Area Displays the calculated percent area for the named peak compared to all named peaks This value results from dividing the individual peak area by the sum of all named peak areas for the capil lary and multiplying by 100 shown for named peak sample data only Width Displays the calculated peak width shown for sample data only S N Displays the calculated signal to noise ratio for the peak shown for sample data only Capillaries Pane User Specified Peak Names Click the Capillaries tab to view tabulated results for sample proteins associated with specific primary anti bodies in the run data Compass labels these sample peaks automatically using user defined peak name set tings Each row in the table shows the individual results for the named peaks detected in each capillary Data for samples in the capillaries table is shown in the following example HEE Peaks Capillaries al
102. 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 r al 8888828 8 8 Chemiluminescence IE Graph l Image Lane 7 ge O co TEF Sample sN F Capillaries biotin lad biotin lad biotin lad biotin lad biotin lad biotin lad Primary Blocki Blocki Blocki Blocki Blocki Blocki Name Position Ldr 12 Ldr 40 Ldr 66 Ldr 90 Ldr 116 Ldr 180 381 607 718 756 792 870 MW kDa 12 40 66 90 116 180 Height 670 1 750 3 712 9 1507 7 974 4 581 3 Area Width 221 21 0 18 3 17 1 18 0 24 5 Data in this view is for immunodetected sample proteins only Graph view data displays electropherograms in chemiluminescence units y axis and molecular weight in kDa x axis Lane view data displays immunodetected sample proteins only Image view data displays immunodetected sample proteins only Results for each immunodetected protein are shown in the peaks and capillaries tables Compass Software for Peggy User Guide page 148 Chapter 8 Size Assay Data Analysis NOTE The reported molecular weight for immunodetected sample proteins in Compass may vary slightly from predicted molecular weights based on sample and assay conditions For information on checking and identifying sample peaks see Step 4 Checking the Ladder on page 169 or Step 5 Checking Samples on page 171
103. 2 K562 anti ERK1 2 1 3 cs D3 ES F3 wf K562 anti ERK1 2 1 4 c4 D4 E4 F4 wf K562 anti ERK1 2 1 5 C5 D5 E5 FS wf K562 anti EREL 2 1 6 C6 DG E6 FG K562 anti ERKL 2 1 T C DT E7 F7 K562 anti ERK1 2 1 8 E p E8 Fa K562 anti ERKL 2 1 g co Do EQ FQ K562 anti EREL 2 1 10 c10 D10 E0 F10 K562 anti ERK1 2 1 11 cil D11 H1 F1 RTU K562 anti ERK1 2 1 12 qi Dil El F2 To view all columns Click the Experiment tab then use the scroll bar or click Maximize in the upper right corner To resize columns Click the Experiment tab Roll the mouse over a column border until the sizing arrow appears then click and drag to resize Experiment pane column descriptions for the Peggy Size default assay are as follows Sample Sample name If sample names were entered in the assay template Assay screen those names will display here Otherwise Sample default name will display NOTE Data notification icons will display in the sample column if Compass detected a potential analysis issue or data was manually modified by the user For more information see Compass Run Data Notifica tions and Warnings on page 162 e Primary Primary antibody name If primary antibody names were entered in the assay template Assay screen those names will display here Otherwise Primary default name will display e Cycle Run cycle number There are 12 capillaries in one cycle Compass Software for Peggy User Guide page 140 Chapter 8 Size Assay Da
104. 2 C13 9 Low phos ERKL 2 Cl 4 4 High pho 1 5 s ERKI C1 6 J To view peak area in the peak name columns default Deselect in the upper right corner of the pane This displays calculated peak area for the individual peak only Compass Software for Peggy User Guide page 278 Chapter 9 Charge Assay Data Analysis Viewing Run Data Each run file contains the following data for up to 96 capillaries Sample data For the immunodetected proteins in the sample Standards data For the fluorescent standards run with each sample Registration data For tracking capillaries as they are moved for various assay steps Data can be viewed for one all or individually selected capillaries Switching Between Sample Standards and Registration Data Views You can switch between viewing sample standards and registration data in a run file using the View bar or View menu B Instrument Window Single View Multiple View Standards Registration Samples Filter View Region Show Hidden Data buttons in the View bar Show Standards Show Registrations Show Samples Compass Software for Peggy User Guide Viewing Run Data page 279 To view sample data Click Show Samples in the View bar or select View in the main menu and click Samples DemoData_Charge_HeLa_ERK12 Compass File Edit View Instrument Window Help u E e Lo i ames Assay C3 Run Summary Sample High High
105. 2012 10 26 09 37 2012 10 24 06 54 2012 10 22 10 44 2012 10 21 10 43 2012 10 02 21 50 2012 07 23 19 26 2012 05 24 23 12 2012 03 31 07 00 3 Select a log file and click View The log details will display Compass Software for Peggy User Guide View Chapter 7 Controlling Peggy Viewing Log Files 2011 09 25 10 19 30 793 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 19 35 860 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 E 2011 09 25 10 19 40 878 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupP WM 8 2011 09 25 10 19 45 896 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 19 50 911 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 19 55 925 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 00 941 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 05 957 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 10 971 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 15 987 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 20 21 003 temperature INFO SetPoint C 23 0 Chamber C 24
106. 259 Importing and Exporting Analysis Settings 261 Importing Analysis Settings 00 ccc cee 261 EXDOMING ANGIVSIS SENGS ajcctereiuveatccues 261 Chapter 9 Charge Assay Data Analysis 263 Analysis SCreCN OVEVIOW oi ciene sd iiir rina i 264 ANINI SEICCN GICs civil cic os ethee nad 264 Software Menus Active in the Analysis Screen 266 COONS ast eat tote ac TEET 268 ODEO G ONE RUD PC ioe Errr 268 OPENI MUUDE RUE ES ci Sn tone nities amp 268 How Run Data is Displayed in the Analysis Screen 271 Experiment Pane Assay and Capillary OM MONO ESEE A uti EAE E 271 Compass Software for Peggy User Guide page v Graph Pane Electropherogram Data 2 2 Image Pane Capillary Separation Image Data 273 Lane Pane Virtual Blot Like Image Data 2 4 Peaks Pane Calculated Results 006 275 Capillaries Pane User Specified Peak Names 276 ewig TAU ID ONO 22a sie taciin cette halen wetiae lucas 2 8 Switching Between Sample Standards and Registration Data Views sic eth 5 teu Guede 2 8 Selecting and Displaying Capillary Data 283 Switching Between Single and Multiple Views of POCO HOC a seats pated ite oor atone oes 287 Hiding Capillary DO orrera ccc ccc cece 291 Setting Run Data Display Filters 292 Compass Run Data Notifications and Warnings 294 CHECKING VOENINCSUIS etary oben ERN EOE ES 295 Step 1 Review the Fluorescent Sizing Standards Ch CRE eee ee eee
107. 3 15AM TY CD PA 3 Fee 6 Lii is z u _ _ os Simri 6 05PM 6 09PM 7 18PM 11 39PM 12 05AM 2 14AM 3 22AM 3 56AM CTY CD PT zT E f r 7 L Dirr 7 19PM 7 23PM 8 32PM 12 20AM 1246AM 2 55AM 4 03AM 4 37AM T CA r 3 Gal po 8 Lii t N aad h k foo ane ore 8 32 PM 8 37 PM 9 46 PM 1 09 AM 1 36 AM 3 44AM 4 53AM 5 26AM v Compass Software for Peggy User Guide Software Overview page 5 Analysis Screen The Analysis screen is used to view run data including electropherograms capillary images lane view data and tabulated results Any post run analysis is also done in this screen 2012 03 05_11 51 19 _HelaControlERKassay Compass fol eis File Edit View Instrument Window Help e 2Gla le 5 cay C Run summary E Experiment a 2 ee Graph 4 Image E Lane 9 m gi D7 A A Sample Primary Cycle biotin Blocki 1 hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki amp
108. 300 200 100 0 H a L pa _ oom Out 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 Rema Pe Hide HE Peaks gt iii HE Capillaries Kame 7 Add Peak Sample Primary Cap Peak Name Position pl Height Area fo Area Widt Add Baseline Point Low p ERKL 2 33 0034 Clear All Copy Ctrl C Compass Software for Peggy User Guide Changing Sample Protein Identification page 315 5 To view hidden peak data click View in the main menu and click Show Hidden Hidden peak data will display in the results table and be marked with an X ppErk1 pErk1 Erk1 ppERK2 Chemilumines cence 400 300 200 100 000 900 200 700 600 Fh 400 300 200 gt 8 pl Sample Primary Cap Peak Name Position p Height ea Area Width 0 0347 6 Tounhide a peak right click on the peak in the electropherogram or peaks table and select Unhide Peak Changing Peak Names for Sample Data If Compass did not automatically name a sample protein associated with a specific primary antibody this can be adjusted manually To do this 1 Click Show Samples in the View bar 2 Click Single View in the View bar 3 Click on the row in the experiment pane that contains the sample you wish to correct then click the Graph tab Compass Software for Peggy User Guide page 316 Chapter 9 Charge Assay Data Analysis 4 Right click the peak in the electrooherogram or peaks table and click Name then click a name in the list Compas
109. 31 Chapter 8 Size Assay Data Analysis Chapter Overview Analysis Screen Overview Opening Run Files How Run Data is Displayed in the Analysis Screen Viewing Run Data Compass Run Data Notifications and Warnings Checking Your Results Group Statistics Copying Data Views and Results Tables Exporting Run Files Changing Sample Protein Identification Changing the Virtual Blot View Changing the Electropherogram View Closing Run Files Compass Analysis Settings Overview Advanced Analysis Settings Images Analysis Settings Peak Fit Analysis Settings Peak Names Settings Standards Settings Importing and Exporting Analysis Settings Compass Software for Peggy User Guide page 132 Chapter 8 Size Assay Data Analysis Analysis Screen Overview The Analysis screen is used to view run data including electropherograms capillary images lane view data and tabulated results Any post run analysis is also done in this screen To access this screen click Analysis in the screen tab E assay C Run Summary Analysis Screen Panes The Analysis screen has six panes each displays the following data for up to 96 capillaries per experimental run Experiment Lists the assay protocol steps and assay template information e Graph Displays electropherogram data for immunodetected sample proteins fluorescent standards or capillary registrations e Image Displays a 12 capillary image of the separated immunodetected sample proteins fluores cent s
110. 38888 O oo s CF ka Fe W M y e O Show named peaks only Only data for the checked capillaries will display in data views and results tables unchecked capillaries will be hidden To filter data to show named peaks only Select View in the main menu and click Filter Select Show named peaks only then click OK Only data for peaks that Compass identified automatically using the user defined peak name analysis parameters will display in data views and results tables all other peak data will be hidden Compass Software for Peggy User Guide page 162 Chapter 8 Size Assay Data Analysis Compass Run Data Notifications and Warnings If Compass detects a potential data issue a notification or warning will display next to the row in the experi ment pane A list of warnings notifications and corrective actions are as follows Manual correction of sample data notification Indicates the user modified sample data manually such as adding or removing a sample peak Rolling the mouse over the icon displays the type of data modification made Primary High ERKI 2 1 J Low p ERKL 2 1 High ERKL 2 Cycle Baseline Manual Standards warning Indicates that one of the standards may not be identified prop 5 Il erly This can be resolved by manually identifying the standards Please refer to Step 2 Checking Fluorescent Sizing Standards on page 164 for details Rolling the mouse over t
111. 5 Page 3 Start The resource tray will automatically open Fill the Wash Buffer Running Buffer and Matrix Removal Buffer cups and insert the capillary box in the primary capillary box position Add a secondary capillary box if more capillaries are required Click Next when finished The resource tray will close NOTE Make sure enough capillaries are loaded to complete the run A capillary box should be inserted in the primary position first and if additional capillaries are needed use the secondary position When the primary box is depleted Peggy will automatically move to the secondary box Discard leftover Running Buffer Wash Buffer and Matrix Removal Buffer prior to refilling bottles for new run Compass Software for Peggy User Guide page 56 Start Run Start Set up the instrument to start a new run Load the Wash Running and Matrix Removal buffers Wash Running Matrix Removal Load a primary cap box Add a secondary cap box if more capillaries required Primary Cap Box Secondary Cap Box Next gt Cancel lt Back Chapter 3 Running a Size Assay on Peggy NOTE You an also refer to the labels on the resource tray for proper insertion of reagents Primary Capillary Box Secondary Capillary Box Compass Software for Peggy User Guide Wash Buffer Running Buffer Matrix Removal Buffer Starting a Run page 57 6 Page 4 Load Sample Plate he sample tray will automatically ope
112. 550 K 00 OK 650 Compass Software for Peggy User Guide Standards Settings page 383 11 To use the new group as the default analysis settings for the run file data click the arrow in the drop down list next to Default then click the new group from the list Analysis settings in the new group will then be applied to the run data Analysis Settings Standard New Standards Default Standard i Standard New Standards 12 Click OK to save changes Changing the Default Standards Group 1 Select Edit in the main menu and click Analysis then click Standards in the options list 2 Click the arrow in the drop down list next to Default then click a new default group from the list Analysis Settings Standard New Standards Default m Standard 3 Click OK to save changes Analysis settings in the group selected will be applied to the run data Modifying a Standards Group 1 Select Edit in the main menu and click Analysis then click Standards in the options list 2 Click on the group in the analysis settings box you want to modify Compass Software for Peggy User Guide page 384 Chapter 9 Charge Assay Data Analysis Analysis Settings Standards New Standards 3 Modify fluorescent standards information as described in Creating a New Standards Group on page 380 4 Click OK to save changes The new analysis settings will be applied to the run data Deleting an Analysis G
113. 9 LookupPWM 8 2011 09 25 10 22 41 463 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 22 46 480 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 T 4 Click Save File As to save a copy of the log file Compass Software for Peggy User Guide page 127 page 128 Chapter 7 Controlling Peggy Self Test Logs 1 Select Instrument in the main menu and click Properties to display system properties 2 Click View Test Log A list of self test logs will display PLOOD Self Tests Test Date Status 2012 10 23 14 33 20 PASSED 2012 10 11 13 11 50 PASSED 2012 10 10_16 22 29 FAILED 2012 10 10 16 15 12 FAILED 2012 10 10 14 25 48 STOPPED 2012 10 10 14 24 41 FAILED 2012 10 05 14 05 02 PASSED 2012 10 05 14 02 32 PASSED 2012 10 05 13 59 20 PASSED 2012 06 19 16 05 22 PASSED Compass Software for Peggy User Guide Viewing Log Files J Dark Masters Camera Fan Temp Sensors Level Filter Not In Filter Not Out Filter Out Filter In LED Low Not On LED Low On LED Not On LED On Voltage On Lid Remover Not Down Lid Remover Not Up Lid Remover Up Lid Remover Down Pipet Not Down Pipet Not Up Pipet Up Pipet Down Duration sec 0 024 11 720 0 056 0 161 0143 0 163 0 593 0 665 0 025 0 017 0 016 0 016 8 965 0 174 0 147 0 468 0 309 4 Click Save File As to save a copy of the log file Compass Software fo
114. Ae NS T T enea a E R 3 PE SUN SCI CON eei ni Mara aie ted tiarsree 4 PUNGIYSIS SCHCC Os e e ain daonh immune J A A Oe EEEE EE T EEEE 5 TO BON eriat renn O Er OERE 6 MTE eeen Er ely nA STT 6 MEOE USBI ERSEN 6 SERET A O E EE E EE Z VICVADGl eevee ARR ieee eet Olde Aa EEA f COMBAS US PO er EEE OE ethene 7 SOM WV AICI CIES rases EENS 7 UIC E EE AENA EEE EEE tree ee 8 OIA E E E EEA 8 Veve VI CTU ee E EAS 8 TUNEN CME eee mene Monee etre eee eee eee 9 ETONE EE EE EEE AE 9 POH VIMO AAE EEEE tas cute 10 Changing the Compass Main Window Layout 10 Resizing the Main Compass Window 10 RESIZING TG SCICCWIGD os ds tame tcete rece sas a 10 Compass Software for Peggy User Guide page i RESIZING SCLCCIT PF ONCS a oie ei eect nae adacar atic I Changing the Location of Screen Panes 2 Restoring the Main Window to the Default LOYO noc he Racha E AAN 14 DO TE A ee ra eA EAR 14 Checking for and Installing New Versions of RO EE EN P SENE E AE 14 VIEWING ROICUSC NOLES Terst E SE 15 Viewing the Software LOG 6c sE a cece eee 15 Compas Verion INIONMOQNO na coniavdvecdseesasewes ies Drect GNG File INTONMOUON oyrna s RELEE ri 16 E ares ial E ate A EE 18 Chapter 2 Size Assays 19 PSSOY STEEN OVE E Wo sissen rira a Wale aaia 20 ASSY STEN PONOS 4 hide EE AERE 20 Software Menus Active in the Assay Screen 21 keagent GOOF COGIAG aara Er O EEEN T 23 ORNO GTA S O AEA e A 24 CTEGUAG GINCW ASS OY aaneen aa i dente
115. Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blacki Sample i Name Position MW kDa Height biotin lad Blocki 1 Ldr 12 413 12 522 8 biotin lad Blocki Ldr 40 629 40 699 7 biotin lad Blocki Ldr 66 739 66 682 5 biotin lad Blocki Ldr 90 776 90 1598 4 biotin lad Blocki J pe pita biotin lad Blocki ec gt A F amp F FP FP PW WwWWHsnwnwnwnwnwnwnwnwwnwnwnnnn ns Nn NNN N NN FP RP RP RR RP RR eR ee nw amp Ww Nh NOTE The reported molecular weight for immunodetected sample proteins in Compass may vary slightly from predicted molecular weights based on sample and assay conditions Compass Software for Peggy User Guide page 134 Chapter 8 Size Assay Data Analysis Software Menus Active in the Analysis Screen The following software menus are available File Edit View Instrument when Compass is connected to Peggy Window Help The File Edit and View menu options specific to the Analysis screen are described next File Menu The following File menu options are active Open Run Opens a run file Edit View Instrument Open Run i Add Run i Close Close All Save Save As Export Tables Export Spectra k Exit Add Run Opens and views other run files in addition to those that are already open Close Closes the run file currently being viewed Close All Closes all open run
116. Compass Administrator A successful log in will display the Compass main window with the user information in the lower status bar The full user name is displayed with the unique user ID in parenthesis File Edit View Instrument Window Help Primary Cap Peak Name Position MW kDa Height Area Area Width S N B Compass Software for Peggy User Guide Saving Changes page 405 If there is no activity in Compass for 20 minutes the user must re enter their password to perform any con trolled actions Inactivity timeout please re enter password User steveg Resolving Log In Issues Log in failures may occur when The server is temporarily unavailable Compass is using the wrong IP address When this happens the following message displays Cannot connect to server 10 1 2 181 2000 Launch Compass with Access Control disabled Compass will not open controlled files Click Disable to restart Compass with Access Control disabled Verify or correct the server IP address then close and restart Compass to log in with Access Control enabled Saving Changes When Save is selected from the File menu a dialog box will display to allow you to enter a comment before saving the signed file Compass Software for Peggy User Guide page 406 Chapter 11 Compass Access Control and 21 CFR Part 11 Compliance Comment Changed Baseline from 1 0 to 2 0 The comment is added to the signa
117. Compass Assay File Simple Western assay 9 29 2011 3 18 PM Compass Assay File Erk Assay assay 9 12 2011 1 11 PM Compass Assay File Simon Decontamination Procedure assay 9 12 2011 1 11 PM Compass Assay File Generic Simple Western 9 9 2011 assay 9 9 2011 7 18 PM Compass Assay File Simple Western Demo Plate assay 9 9 2011 7 18 PM Compass Assay File HE Desktop Libraries ES Documents E My Documents File name My New Assay a ach Hide Folders NOTE New assays are saved in the Compass Assays directory Compass Software for Peggy User Guide Creating a New Assay page 85 Step 8 Modify Default Analysis Parameters Optional You can preset the parameters used to analyze run data generated with the assay 1 Select Edit from the main menu and click Default Analysis The following screen will display Advanced Images Analysis Settings Analysis Settings Advanced Peak Fit Advanced E ie Peak Names Standards Standards Peak Width Allowable Drift Sample m Peak Fit Starting Width Ratio Remove PEAN Image dii cvencest Median Filter Threshold Ratio oak Median Filter Threshold Limit Remove Restore Original 2 ProteinSimple recommends using the default parameters for Simple Western assays However users can modify any parameters as needed then click OK For detailed information on analysis parameters please refer to Compass Analysis Settings Overview on page 219 Compass Software for Peg
118. Compass identified automatically using the user defined peak name analysis parameters will display in data views and results tables all other peak data will be hidden Compass Software for Peggy User Guide page 294 Chapter 9 Charge Assay Data Analysis Compass Run Data Notifications and Warnings If Compass detects a potential data issue a notification or warning will display next to the row in the experi ment pane A list of warnings notifications and corrective actions are as follows Manual correction of sample data notification Indicates the user modified sample data manually such as adding or removing a sample peak Rolling the mouse over the icon displays the type of data modification made Primary High ERKI 2 1 J Low p ERKL 2 1 High ERKL 2 Cycle Baseline Manual Standards warning Indicates that one of the standards may not be identified prop 5 Il erly This can be resolved by manually identifying the standards Please refer to Step 2 Checking Fluorescent Sizing Standards on page 296 for details Rolling the mouse over the icon displays warning details Sample Primary 5 2 Sample Primar 5 3 Standards Warn ing Low Confidence Manual correction of standards data notification Indicates the user modified the standards data manually Rolling the mouse over the icon displays the type of data modification made Qo Registrations warning Indica
119. ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 zNa kb e e e e e i i i i i i i a a a M M a a I A G lt mn ke e e e e a a i ia E Graph 2 Image F7 Lane IIE ERRE PSS PLL LL PLS Ss E S d FERE ii SLEPEPLPEEP EEL EDEL DED K x CEPEPEEEEE EEL SF MSH FM I 2 3 ote s FH Capillaries Sample a Cap Peak Name Position Height Area Area Width S N a ii nii 3 High CEI 624 0 0239 1538 7 Compass Software for Peggy User Guide Viewing Run Data page 287 Switching Between Single and Multiple Views of the Capillaries You can switch between displaying run data in the graph image and lane panes in a per capillary single format or up to 96 capillary format This view is selected in the View bar or the View menu geerzEl E Instrument Window Single View Multiple View Standards Registration Samples Filter View Region Show Hidden Capillary view buttons in the View bar E Single View Multiple View Compass Software for Peggy User Guide page 288 Chapter 9 Charge Assay Data Analysis To view data ina per capillary format Click Single View in the View bar or select View in the main menu and click Single View Eye DemoData_Charge_Hela_ERK12 Compass o fees File Edit View Instrument Window Help bas Ee fg E Assay TE Run Summary Sample Primary Cy
120. ERK2 ERK2 1500 ppERK2 300 Hi phospho HeLa 200 C13 L sgegezues Chemilumines cence o 8 50 51 52 53 54 55 56 57 58 59 GD Gi G2 6 amp 2 G4 65 6E Gr ES EJ Z0 pl Plot Labels You can customize the plot labels displayed on the electropherogram with these options Plot Label Wl Sample M Attribute El Primary E Attribute W Capillary Z Exposure Plot labels are shown on the right side of the graph pane Sample Checking this box will display the sample name If sample names were entered in the assay template Assay screen those names will display here Otherwise Sample default name will display Primary Checking this box will display the primary antibody name If primary antibody names were entered in the assay template Assay screen those names will display here Otherwise Primary default name will display Capillary Checking this box will display the cycle and capillary number For example C1 3 indicates cycle 1 capillary 3 Attributes Checking this box will display attribute text If attribute information was entered for sam ples or primary antibodies in the assay template Assay screen they can be selected as plot labels Compass Software for Peggy User Guide page 338 Chapter 9 Charge Assay Data Analysis Exposure Checking this box will display the exposure time s used for the data The fo
121. Enter a test message and click OK Compass Software for Peggy User Guide Setting Peggy Up to Send Tweets page 397 Sending Tweet Message Message to send to Twitter Compass lest Message at 20120605 170630 6 Ifthe test Tweet was successful Compass will display the following message 7 Click OK to save changes and exit Peggy will automatically tweet as the selected options occur as shown below Compass Software for Peggy User Guide page 398 Chapter 10 Setting Custom User Preferences r Home CD Connect F Discover a PL0006 Edit your profile Test dive Gel Free Hands Free Blot Free Santa Clara http Awww proteinsimple com Orc 0 Tweets gt Tweets Following PLOOOG6 Followers Compass Test Message at 20120615 201241 Favorites Lists PLOOOG Tes I Compass Test Message at 20120615 201146 PLOOOG6 Test Hi Thanks for the free time GREAT Data View conversation PLOOO6 Compass Test Message at 20120605 170630 Changing the Twitter Account To change the Twitter account Peggy uses 1 Select Edit in the main menu and click Preferences and click Twitter in the preferences list 2 Click Clear 3 Set up the new account as described in Setting Peggy Up to Send Tweets on page 394 Compass Software for Peggy User Guide Setting Peggy Up to Send Tweets page 399 Sending Manual Tweets from Peggy You can send tweets directly from Peggy For example you may want to notify o
122. N NOTE To clear the manual addition or removal of baseline points and go back to the original view of the data right click in the electropherogram and click Clear All Selecting the X Axis Molecular Weight Range The molecular weight range used for the x axis can be changed To do this Select View in the main menu and click View Region The following pop up window will display E Full Range Lower 1 0 Upper Ce Compass Software for Peggy User Guide page 218 Chapter 8 Size Assay Data Analysis Tochange the x axis MW range displayed for the run data Enter new values in the Lower or Upper range in molecular weight kDa and click OK Electropherogram and virtual blot data will update to display only the data in the entered range To see the full x axis MW range included in the run data Check Full Range Electrooherogram and virtual blot data will update to display the full range of available data E Graph BS Image Lane Be i m Samples 13004 ERK2 12004 1100 1000 K562 C1 10 900 2 7 E coo 500 5 400 ERK TF 300 200 j 100 1 100 4 i 12 40 6 90 116 180 MW kDa NOTE You can change the default x axis range that Compass uses For more information see Peak Fit Analysis Settings on page 231 Closing Run Files If more than one run file is open you can close just one file or all the open files at the same time Toclose one
123. Overlay on page 392 Compass Software for Peggy User Guide page 328 Chapter 9 Charge Assay Data Analysis Overlaying Multiple Electropherograms The electrooherogram data for multiple capillaries can also be overlaid on top of each other for comparison in the graph pane To do this 1 Click Single View 2 Select multiple rows in the experiment pane 3 Click the Overlay the Plots button The individual electropherograms for each row selected will overlay in the graph pane K562 C1 8 K562 C1 10 K562 C1 11 D TI T TI uw a E 3 E T t O 8 2 amp MW kDa Users can customize the colors used for the overlay plot display For more information see Selecting Custom Plot Colors for Graph Overlay on page 392 Compass Software for Peggy User Guide Changing the Electropherogram View page 329 Zooming To zoom in on a specific area of the electropherogram hold the mouse button down and draw a box around the area with the mouse IE Graph gt 1 Image E Lane H i r coc b Hi phospho HeLa C22 L Chemiluminescence 300 200 100 D0 900 a00 d 700 600 500 4 400 4 300 200 4 SE a0 5i 52 53 54 55 56 S7 58 59 S0 pl Compass Software for Peggy User Guide page 330 Chapter 9 Charge Assay Data Analysis To return to default scaling right click in the electrooherogram and click Zoom Out l b E Image Lane T BF Fo A H
124. PM 8 37 PM 9 46 PM 1 09AM 1 36AM 3 44AM 4 53AM 5 26AM v Software Menus Active in the Run Summary Screen The following software menus are available e Instrument when Compass is connected to Peggy File Edit Window e Help Compass Software for Peggy User Guide page 108 Chapter 6 Run Status The File and Edit menu options specific to the Run Summary screen are described next File Menu The following File menu options are active Edit View Instrument Open Run k Add Run F Close Close All Save Save As Export Tables Export Spectra j Exit e Open Run Opens a run file Add Run Open and view other run files in addition to the one that is already open e Close Closes the run file currently being viewed e Close All Closes all open run files Exit Closes Compass Edit Menu The following Edit menu options are active Instrument Window He Cut Copy Ctrl C Paste Ctrl V Analysis Preferences Preferences Set and save custom preferences for data export plot colors in the graph and Peggy s Twitter settings See Chapter 10 Setting Custom User Preferences for more information Compass Software for Peggy User Guide Opening Run Files page 109 Opening Run Files You can open one run file or multiple run files at a time to compare information between runs Opening One Run File 1 Select File in the main menu and click Open Run Edit Ins
125. Portable Document Format PDF 4 Select a directory to save the file to and enter a file name then click OK Exporting Run Files Results tables and raw plot data can be exported for use in other applications Exporting Results Tables To export the information in the peaks and capillaries tables 1 Click File in the main menu and click Export Tables 2 Selecta directory to save the files to and click OK Data will be exported in txt format NOTE To exclude export of standards data or export results table data in csv format see Setting Data Export Options on page 391 Exporting Raw Sample Electropherogram Data To export raw sample plot data 1 Click File in the main menu and click Export Spectra Compass Software for Peggy User Guide Changing Sample Protein Identification page 183 Edit View Instrument Window Help Open Run p Add Run O kx Graph Image Close Close All Save 5500 4 Save s i 5000 Export Tables t pee E Export Spectra j Text Format Exit a To export data in txt format Select Text Format Plots will be exported in one file for all capil laries To export data in cdf format Select Andi Format Plots will be exported in one file per capil lary 2 Select a directory to save the files to and click OK Data will be exported in the selected format Changing Sample Protein Identification Compass allows you to customize what sample proteins
126. Primary E Attribute Capillary Exposure You can customize the labels used to identify peaks in the electrooherogram with these options TT Matching Peak Names Peak Names Peak Values Matching Peak Names Checking this box will draw vertical lines through each named peak Using this option with Stack the Plots or Overlay the Plots features is useful for visual comparison of named peaks across multiple capillaries Compass Software for Peggy User Guide Changing the Electropherogram View page 203 ks Graph BA Image Lane t GF i imi 5 8 g 3 8 8o ee 2 a o T o nw a E a E T E O o E aE i nw a i E a E Q i a O T T a T nw a i E a E T Lo oc i MW kDa Peak Names Checking this box will display peak name labels on all named peaks in the electrophe rogram NOTE If more than one peak label option is selected peak name labels will always be used for named peaks Compass Software for Peggy User Guide page 204 Chapter 8 Size Assay Data Analysis Chemilumines cence 000 200 200 700 600 500 400 300 200 100 0 40 MW kDa e Peak Values Checking this box will display the molecular weight labels on all peaks in the electro pherogram NOTES When viewing standards and registration data this option is called Peak Positions Labels displayed are peak positions rather than molecular weight If
127. RK1 2 Low phosp ERK1 2 High phos ERK1 2 Low phosp ERK1 2 High phos ERK1 2 Low phosp ERK1 2 High phos ERK1 2 Low phosp ERK1 2 High phos ERK1 2 Low phosp ERK1 2 High phos ERK1 2 Low phosp ERK1 2 oy S ee Erki pl 5 90 Area 12533 HE Peaks _ F Capillaries Sample Primary Cap Peak Name Position i Area Area Width S N High ERK1 2 Gih 1 ppErkl 499 j 6337 58 7 0 0464 5179 High ERK1 2 Gih 2 pErkl 583 1411 131 00265 3211 High ERK1 2 C11 3 ppERK2 607 5864 62 4 0 0239 1538 7 High ERK1 2 C1 1 4 Erk 3054 28 3 oaa 879 5 5 6 n n oe m 159 2 3529 Step 5 Assigning Peak Names Optional Compass can identify and automatically name sample proteins associated with specific primary antibodies in the run data using user specified peak name settings For more information on how to do this see Peak Names Settings on page 367 Compass Software for Peggy User Guide page 304 Chapter 9 Charge Assay Data Analysis Group Statistics The Grouping View is used to analyze replicates by calculating the mean standard deviation CV and SEM for named proteins see Peak Names Settings on page 367 for detailed information on entering named proteins Statistics for each protein are also plotted for easy comparison between samples antibodies and proteins Using Groups 1 A group is automatically created for capillaries with the same sampl
128. Ready Cleanu capillaries from system trays This is required to prepare the system for the next run 2 Click Cleanup Cleaning Cleaning Stop Wed 3 48 PM Wed 3 56 PM P Allow Peggy to complete the cleaning protocol which takes about ten minutes When complete instru ment status will change to Ready and a new run can be started Compass Software for Peggy User Guide page 105 Chapter 6 Run Status Chapter Overview Run Summary Screen Overview Opening Run Files Viewing File and Run Status Information Watching Standards Separation Movies Viewing Current and Voltage Plots Switching Between Open Run Files Closing Run Files Compass Software for Peggy User Guide page 106 Chapter 6 Run Status Run Summary Screen Overview The Run Summary screen is used to monitor run progress watch movies of the fluorescent standards sepa ration and view current and voltage plots for a run To access this screen click Run Summary in the screen tab FA Assay 4 i Run Summary ik Analysis Run Summary Screen Panes The Run Summary screen has three panes Status Displays run file information and current status of a run in progress Separation Lets you view a movie of the fluorescent standards separation for each cycle of the experimental run IV Plot Lets you view plots of the total current and voltage measured during separation for all 12 capillaries for each cycle of the experimental run Compass So
129. Roll the mouse over a column border until the sizing arrow appears then click and drag to resize Peak table column descriptions are as follows Sample Sample name If sample names were entered in the assay template Assay screen those names will display here Otherwise Sample default name will display e Primary Primary antibody name If primary antibody names were entered in the assay template Assay screen those names will display here Otherwise Primary default name will display e Cap Cycle and capillary number For example C1 3 indicates cycle 1 capillary 3 NOTE Peggy runs up to eight cycles in an experiment 12 capillaries at a time Capillary and cycle numbers are displayed in the Experiment tab Peak Peak number Peaks are numbered in order of detection Compass Software for Peggy User Guide page 276 Chapter 9 Charge Assay Data Analysis Name Peak name Displays peaks that Compass named automatically using the user defined peak name analysis parameters Cells will be blank if Compass was not able to name the peak or if naming parameters were not entered Position Displays the pixel position of a peak in the image pl Displays the calculated pl for the peak Height Displays the calculated peak height Area Displays the calculated peak area Area Displays the calculated percent area for the named peak compared to all named peaks This value results from dividing the individual peak ar
130. Soak Time sec Secondary Ab Time min 150 0 60 0 150 0 60 0 150 0 60 0 150 0 60 0 Well Row A a ee a E So 2 2 2 2 2 2 2 2 Washes Wash Soak Time sec Detection Well Row Detection Profile jl Jl jl jl jl jl jl 6 Exposures 6 Exposures 6Exposures 6 Exposures 6Exposures 6 Exposures 6 Exposures 6 Exposures page 31 2 Five incubation steps are allowed per protocol Users can select the type of incubation for each step The available incubation types and their default Simple Western use is as follows To change the type click the incubation step name and select an option from the drop down list First incubation Blocking Second incubation Primary antibody Third incubation Secondary antibody Fourth incubation User defined tertiary antibody Fifth incubation User defined custom Compass Software for Peggy User Guide Blocking page 32 Chapter 2 Size Assays 3 If needed change the primary incubation time To do this click the cell in the value column next to Pri mary Ab Time min and enter a new value in minutes Cyclel Cycle 2 Cycle 3 Cycle 4 Cycle5 Cycle 6 Cycle 7 Cycles Separation Matrix Stacking Matrix Sample Separation Time min 40 0 40 0 40 0 40 0 40 0 40 0 40 0 40 0 Separation Voltage volts 250 250 250 250 250 250 250 250 Matrix Removal Blocking Time min 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 Primary Ab Time rin 120 0 120 0 120 0 120 0 12
131. Software for Peggy User Guide Advanced Analysis Settings page 227 Cycle 1 8 When this option is selected group settings will be applied to all capillaries in the cycle Custom settings Lets you choose specific capillaries to apply the group settings to When this option is selected the following pop up box appears to let you enter a specific capillary number or range of capillaries Enter cycle and capillary descriptor Examples 2 3 1 1 12 Cycle 2 capillary 3 and cycle 1 capillaries 1 through 12 1 3 6 8 10 Cycles 1 through 3 capillaries 6 8 and 10 6 Ifyou need to change the analysis group used for a data set click the cell in the Settings column and click the down arrow Select a group from the drop down list Override Settings Advanced Advanced 7 Repeat the previous steps to apply other groups to specific run data 8 To remove a data set click on its cell in the Apply To column then click Remove 9 Click OK to save changes Compass Software for Peggy User Guide page 228 Images Analysis Settings Chapter 8 Size Assay Data Analysis The Images analysis settings page lets you see what chemiluminescent exposures were taken during the run and view data for different exposures in the Analysis screen To access these settings select Edit in the main menu and click Analysis then click Images in the options list P Analysis Sally Hela Control ERK Demo Data cbz t
132. Superuser status Designates that this user has all permissions without explicitly assigning them The groups this user belongs to A user will get all permissions granted to each of his her group Hold down Control or Command on a Mac to select more than one Available groups Q Filter Reviewer Operator Scientist Groups Chosen groups Choose all O Remove all Specific permissions for this user Hold dovm Control or Command on a Mac to select more than one Available user permissions Chosen user permissions gt Q Filter User permissions auth auth auth auth auth auth auth auth auth auth auth auth auth Important Date joined Delete user Deny analysis editing user Deny copy export of data user Deny instrument administrati user Deny instrument control user Deny plate editing user Deny protocol editing user Deny sign off of data user Allow analysis editing user Allow copy export of data user Allow instrument administrati user Allow instrument control user Allow plate editing user Allow protocol editing Choose all Remove all 2013 06 11 Today 03 19 34 Now 2013 06 11 Today 03 19 34 Now Save and add another Save and continue editing Save Compass Software for Peggy User Guide page 415 page 416 Chapter 11 Compass Access Control and 21 CFR Part
133. T Exposure3 240 seconds Exposure 60 seconds Exposure 120 seconds 3 240 seconds NOTE The number of exposures taken and exposure times shown were specified in the assay protocol Assay screen prior to executing the run and cannot be modified Changing the Sample Data Exposure Displayed The exposure used for the sample data displayed in the Analysis screen can be changed To do this 1 Select Edit in the main menu and click Analysis then click Images in the options list 2 Click the arrow in the drop down list next to All and select an exposure setting 3 Click OK to save changes and exit Sample data for the exposure selected will display in the Analysis screen Compass Software for Peggy User Guide page 360 Chapter 9 Charge Assay Data Analysis Peak Fit Analysis Settings The peak fit analysis settings page lets you view and change peak fit settings for sample data To access these settings select Edit in the main menu and click Analysis then click Peak Fit in the options list NOTE Settings can be modified in an assay prior to starting a run or in a run file once the run has finished executing Analysis changes made to an executing run will not be saved to the final run file Analysis Training data_Hi Low ERK cbz type filter text Peak Fit Advanced as Images Analysis Settings Analysis Settings Peak Fit Peak Fit Peak Names Standards Minimum Peak Fit Range Maximum Baseline Threshold Rem
134. TUIMENT COMMUNION LOG ose cin Eee ena 407 S PEE E EEEE A A TTT 409 Troubleshooting Problems and Suggested SOUU eere a ali E 409 AOO ZAO SCIVE e EAN 410 SCM Cr AGIMUNISTQUON ea aaa 411 Adding Non ddmin Users sssreesei ccc eens 412 Adding AC INANISOUS cub scsadet EnA ea 417 Resetting User PASSWOIS eeraa e eiea 418 FIG O BDE EEEE EE 419 Compass Software for Peggy User Guide page vii page viii Compass Software for Peggy User Guide page 1 Chapter 1 Getting Started with Compass Chapter Overview Launching the Software Software Overview Software Menus Changing the Compass Main Window Layout Software Help Checking for and Installing New Versions of Compass Viewing Release Notes Viewing the Software Log Compass Version Information Directory and File Information Compass Software for Peggy User Guide page 2 Chapter 1 Getting Started with Compass Launching the Software e To open Compass double click the icon on the computer desktop Compass Software Overview Compass has three main screens Assay For creating and reviewing assays Run Summary For viewing run status Analysis For viewing experimental data When Compass opens the Analysis screen displays by default Compass xs File Edit View Instrument Window Help E Assay Run Summary EE Analysis Ready New Run SEs ESE af amp pl E Experiment E Graph 2 Image Lane 3d BF iid i Samp
135. Well Row Load Time sec Washes Wash Load Time sec Wash Soak Time sec Compass Software for Peggy User Guide Creating a New Assay page 77 New cycle columns will display using the same parameters used for cycle 1 Add Remove Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle 8 t Sample b Separation b Immobilization Time sec 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 Ez Primary Ab Time min 120 0 120 0 120 0 120 0 120 0 120 0 120 0 120 0 Secondary Ab Time min 60 0 60 0 60 0 60 0 60 0 60 0 60 0 60 0 EE Tertiary Ab Time min 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 b Detection Repeat steps 1 8 to change parameters for the added cycles As cycles are added and reagent locations are selected cycle number assignments will update in the Layout pane assay plate map o Al A h B Le D E F G H l J K L Mi H a P NOTES For more information on changing protocol step parameters other than incubation times contact ProteinSimple Technical Support When a protocol is being edited an asterisk will appear in the Protocol tab to indicate changes have been made and should be saved Step 4 Add Assay Notes Optional 1 Click on the Notes tab 2 Clickin the pane and type any assay or protocol notes This information will be stored with the assay and run data Compass Software for Peggy User Guide page 78 Chapter 4 Charge Assays Protocol TF Notes e This is o
136. When the primary box is depleted Peggy will automatically move to the secondary box Discard leftover Running Buffer Wash Buffer and Matrix Removal Buffer prior to refilling bottles for new run Compass Software for Peggy User Guide page 100 Chapter 5 Running a Charge Assay on Peggy Start Run a _ Start Set up the instrument to start a new run Load the Wash buffer Anolyte and Catholyte Wash Anolyte Catholyte Load a primary cap box Add a secondary cap box if more capillaries required Primary Cap Box Secondary Cap Box Cancel NOTE You an also refer to the labels on the resource tray for proper insertion of reagents Primary Capillary Box Secondary Capillary Box Compass Software for Peggy User Guide Wash Buffer Anolyte Catholyte Starting a Run page 101 6 Page 4 Load Sample Plate he sample tray will automatically open Place the lidded 384 well plate on the cooling block in the tray ensuring that the A1 well position is aligned with the upper left corner of the cold block When this is complete click Next The sample tray will close Start Run Load Sample Plate Remove the old sample plate and load the new plate 1234567 8 91011 12131415617 81921224 wozezrxc xranmoomD A1 Position Compass Software for Peggy User Guide page 102 Chapter 5 Running a Charge Assay on Peggy NOTES Peggy requires that pl
137. a Blocki 1 HeLa Blocki 1 HeLa Blocki 1 HeLa Blocki 1 Biotin Blocki 2 HeLa Blocki 2 HeLa Blocki 2 HeLa Blocki 2 HeLa Blocki 2 HeLa Blocki 2 HeLa Blocki 2 HeLa Blocki 2 HeLa Blocki 2 HeLa Blocki 2 HeLa Blocki 2 HeLa Blocki 2 Biotin Blocki 3 HeLa Blocki 3 HeLa Blocki 3 HeLa Blocki 3 x Hela Blocki 3 Hela Blocki 3 iii Peaks N 2 3 Capillaries m HeLa Blocki 3 Hela Blocki 3 Sample Primary Cap Peak Name Position MW kDa Height Area Area Width HeLa Blocki 3 HeLa Blocki 3 HeLa Blocki 3 HeLa Blocki 3 Biotin Blocki 4 HeLa Blocki 4 HeLa Blocki C1 8 3 967 246 437 7 6272 13 5 Hela Blocki 4 Hela Block S sTaT3 767 87 31346 74540 1000 223 HeLa Blocki 4 HeLa Blocki 4 i 4 Ea ee x 4 iil p Compass Software for Peggy User Guide page 154 Chapter 8 Size Assay Data Analysis To look at data for all capillaries Hold the Shift key and select the first and last rows in the experi ment pane Data for all rows will display in all data views and results tables The following example shows standards data displayed in the lane and peaks panes when all rows are selected The pane scroll bars can be used to view all 8 cycles F Sally HUT78 Screen Compass File Edit View Instrument Window Help vEt 2 0 g E Assay C Run Summary E Experiment x A ie Graph R3 Image E La
138. a list in the same format shown in the Protocol pane Compass Software for Peggy User Guide page 90 Chapter 4 Charge Assays Copying an Assay Template 1 Click on the Template tab 2 Select Edit in the main menu and click Copy 3 Open a document Word Excel etc Right click in the document and select The assay template will be copied into the document as an image exactly as it is shown in the Template pane Printing Protocols and Templates The information in the Protocol pane can be printed as can a graphic image of the information in the Tem plate pane Printing an Assay Protocol 1 Click on the Protocol tab 2 Select File in the main menu click Print and then click Print Protocol Edit Instrument Window Help New Assay k Open Assay Fro Save Save As Import Protecal Import Template Export Protocol Export Template Print Protocol Print Frint Template Exit All assay protocol steps and parameters will be printed as a list in the same format shown in the Protocol pane Printing an Assay Template 1 Click on the Template tab 2 Select File in the main menu click Print and then click Print Template Compass Software for Peggy User Guide Importing and Exporting Protocols and Templates page 91 Edit Instrument _ Window New Assay k Open Assay l apm Pro Save Save As Import Protocol Sa Se Export Protocol Irr Pr Import Template Export Template
139. aA Kg Graph BA mage EJ Lane Eg Be Sample High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 Chemmibminescence BABB Chemibaminescence Cheribminescence Cherribminescence sN i Capillaries Sample Primary Cap Peak Name Position Height Area Area Width S N i i i li a a il Compass Software for Peggy User Guide Viewing Run Data e Tolook at data for multiple sequential capillaries Hold the Shift key and select the first and last rows you want to view in the experiment pane Data for only the selected rows will display in all data views and results tables The following example shows standards data displayed in the image and peaks panes when multiple sequential rows are selected page 285 DemoData_Charge_HeLa_ERK12 Compass File Edit View Instrument Window Help git 2 E H gt E Assay PE Run Summary Analysis E Experiment O Ge Graph 5 Image Lane m Sample Cycle 1 Samples Exp 240 0 a High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phos
140. an Assay Template 1 Click on the Template tab 2 Select File in the main menu click Print and then click Print Template Compass Software for Peggy User Guide page 48 Chapter 2 Size Assays Edit Instrument Window Help New Assay j Open Assay k aem Pro Save Save As Import Protecal Sa Se Irr Pr 5E Import Template Export Protocol Export Template Print p Print Protocol Frint Template Exit The assay template will print as an image exactly as it is shown in the Template pane Importing and Exporting Protocols and Templates The assay protocol in an open assay or run file can be exported as a separate file as can the assay template annotation information This allows the same assay protocol and template information to be imported into another assay at a later time rather than having to re enter assay protocol and template information manu ally Importing an Assay Protocol NOTE Importing an assay protocol imports information into the Protocol pane only 1 Open the assay you want to import the assay protocol in to 2 Select File in the main menu and click Import Protocol 3 Select a protocol file protocol and click OK The imported information will display in the Protocol pane Compass Software for Peggy User Guide Importing and Exporting Protocols and Templates page 49 Exporting an Assay Protocol NOTE Exporting an assay protocol exports information in the Protocol pane only 1
141. an Summary k amp Graph R Image E Lane s D ei BF 2 z aiid 5 A s r z s gt 3D 3D y y y p p p p F gt 3D 3D 9D y 9D p p p p O gt 5d y 9D py 9D p p pp yp yp s gt 5d y y py p p p 9D 92 yp s gt 3d 30 i kDa oE E D O ET ET A AT A LHF AT ET A AI I A T ET ET AT A E A AT ET FHF S A T ET A E AI A A ET ET ET A A T AT ET E A A E 120 1 E _ _ G s i 4 u e Torestore all minimized panes Click Restore on the minimized pane bar ii Reston To restore only one minimized pane Click the pane icon on the minimized pane bar Compass Software for Peggy User Guide page 12 Chapter 1 Getting Started with Compass Torestore a maximized pane to its original size Double click the tab or right click the tab and click Restore Lane Detached Fa Restore M Move p Size a F SF 8 Minimize pd oY Maximize Close To restore all panes to their original sizes Select Window in the main menu and click Default Layout Changing the Location of Screen Panes Panes can be moved to different locations within a screen Tomoveapane Click on its tab and drag it to the new location As the pane is moved area guides will display to assist users in choosing a drop location Area guides with a black arrow let you know that if the pane is dropped at that location it will be resized and relocated as an individual pane in that area of the screen Fy Area
142. andards 4 Click in the first cell in the MW column in the Fluorescent Peaks table New Standards Fluorescent Peaks Position Fit Registration 100 C 200 dl 900 O 5 Enter the molecular weight in kDa for the fluorescent standard Compass Software for Peggy User Guide Standards Settings New Standards Fluorescent Peaks MW Position Fit Registration 3 100 O 12 200 E 180 900 E 6 Click in the first cell in the Position column New Standards Fluorescent Peaks MW Fit Registration 2 12 200 180 900 7 Enter the position of the fluorescent standard peak New Standards Fluorescent Peaks MW Position Fit Registration 2 115 12 200 180 900 Compass Software for Peggy User Guide page 253 page 254 Chapter 8 Size Assay Data Analysis NOTE Standards peak positions are relative to each other Only the difference in their position is used to help identify the standard peaks When entering standard peak information for the first time review the standards data in the Analysis screen to find the correct peak position 8 Repeat the steps above for the remaining standards in the table To add another standard Click Add under the peak table then modify the information in the new row To remove a standard Select its row and click Remove 9 Select which standard should be used for capillary registration by clicking the checkbox in the Registra tion column The first standard is typica
143. anging the Default Analysis Group 224 Modifying an Analysis Group e e aa 225 B E a SE E 225 Applying Analysis Groups to Specific Run Data 225 magas ANGIYSIS SCUTHIAGS iicctnencaewer er dpnaanenes 228 EXODO UE OCCU NOS rena uganate spn wilnyrncen sends 229 Changing the Sample Data Exposure DISDICV CG hciink ink pases E EE EE 230 PEAK FIUAOIVSIS SENINGS 5 cet ii ac om alisha hes 231 kanges eting urere OOS 232 PE AS N I E E E E aes 232 PEOKS E TIG E ynan 232 Peak Fit Analysis Settings Groups 232 Creating ANW PGK FIL GIOUD ei n ee 233 Changing the Default Peak FitGroup 234 Modifying a Pedk Fit GrOUD 0 0 6 c cece ee 233 PRICHNIGIGPCGK FIT GLOU Di a TEE ae 235 Applying Peak Fit Groups to Specific Run Data 235 FCOKINGINGS SONOS Eee EEE nAIRE 238 Peak Names Analysis Settings Groups 239 Creating a Peak Names Group 0 6 05 239 Adding Peak Names Groups 0 60000005 243 Modifying a Peak Names Group 044 244 Deleting a Peak Names GOUD sirener i 244 Applying Peak Names Groups to Run Data 245 sanaa SeT INGS eir Erena 249 Standards Analysis Settings Groups 250 Changing the Capillary Used for the Ladder 250 Creating a New Standards Group 22 Changing the Default Standards Group 257 MOGIVING GStGNGGIds GIOUD 8 icieiae cans 258 Deleting an Analysis Group 0 6 cece eee 258 Applying Analysis Groups to Specific Run Data
144. apter 4 Charge Assays Step 3 Modifying the Assay Protocol Optional 1 Click on the Protocol tab This pane displays the individual steps of the assay protocol and allows you to change parameters as needed When creating a new assay a default protocol will display which auto matically assigns all reagent locations for Cycle 1 Protocol ME Notes ml Cyclel b Sample gt Separation gt Immobilization Time sec 100 0 gt Primary 4b Time min 120 0 gt Secondary Ab Time min 60 0 gt Tertiary Ab Time min 10 0 Detection Each row contains an assay protocol step Each step can contain a reagent assay plate row assignment and one or more parameter settings To view the details for a step click on the white arrow next to the step name ProteinSimple recommends using the default protocol settings for Peggy assays An expanded list of the default protocol step parameters is shown Compass Software for Peggy User Guide Creating a New Assay Protocol 4 Sample Well Row Load Time sec 4 Separation Separation Profile Standards Exposure sec 4 Immobilization Time sec Washes Wash Load Time sec Wash Soak Time sec 4 Primary Ab Time min Well Row Load Time sec Washes Wash Load Time sec Wash Soak Time sec 4 Secondary Ab Time min Well Row Load Time sec Washes Wash Load Time sec Wash Soak Time sec 4 Tertiary Ab Time min Well Row Load Time sec Washes Wash Load Time sec Was
145. are reported in the results tables by making manual adjustments in the electropherogram or peaks table Adding or Removing Sample Data 1 Click Show Samples in the View bar 2 Click Single View in the View bar 3 Click on the row in the experiment pane that contains the sample you wish to correct then click the Graph tab To remove a peak from the data Right click the peak in the electropherogram or peaks table and select Remove peak Compass will no longer identify it as a sample peak in the electro pherogram and the peak data will be removed in the results tables Compass Software for Peggy User Guide page 184 Chapter 8 Size Assay Data Analysis a gY T TI a E 3 E T l D Ssss ESE ES Zoom Out Riwove Peak Hide Name 40 Add Peak MW kDa Add Baseline Point Remove Baseline Point Clear All Copy Ctrl C Carmnle Primary Canillare FRE FRET To add an unidentified peak to the data Right click the peak in the electrooherogram or peaks table and select Add Peak Compass will calculate and display the results for the peak in the results tables and identify the peak in the electropherogram NOTES To remove sample peak assignments that were made manually and go back to the original view of the data right click in the electropherogram and click Clear All Virtual blot data in the lane pane will also update to reflect changes made in the graph pane Compass Software for Peggy Us
146. ars then click and drag to resize Capillaries table column descriptions are as follows Sample Sample name If sample names were entered in the assay template Assay screen those names will display here Otherwise Sample default name will display e Primary Primary antibody name If primary antibody names were entered in the assay template Assay screen those names will display here Otherwise Primary default name will display Capillary Cycle and capillary number For example C1 3 indicates cycle 1 capillary 3 NOTE Peggy runs12 capillaries at a time in a cycle and is able to run up to eight cycles in an experiment The Information on cycle and capillary number is displayed in the Experiment tab e Peak Name Columns An individual column per peak name will display for every peak identified by name in the run data Cells for capillaries in these columns will be blank if Compass did not find peaks automatically using the user defined peak name analysis parameters or none were entered To view area in the peak name columns Select in the upper right corner of the pane This displays the calculated percent area for the named peak compared to all named peaks This value results from dividing the individual peak area by the sum of all named peak areas for the capillary and multiplying by 100 Primary Capillary ppErkl ppERK2 pERK2 High pho ERK1 2 ci Low phos ERKL 2 C1 2 t High pho ERKL
147. asy way to reject indi vidual capillaries from the statistical analysis See Hiding Capillary Data on page 291 for details on how to do this Larger groups of capillaries can also be excluded from analysis To do this select View and click Filter Compass Software for Peggy User Guide Group Statistics page 309 Capillaries o O 0O 0O R A RARA RAOR m ho El Show named peaks only Ce Uncheck the box next to the cycles or capillaries you wish to remove and click OK This data will now be removed from the grouped view statistics Compass Software for Peggy User Guide page 310 Chapter 9 Charge Assay Data Analysis Copying Data Views and Results Tables You can copy and paste data and results tables into other documents or save a data view as a graphic file Copying Data Views 1 Click in the graph or lane pane 2 Select Edit in the main menu and click Copy or right click and select Copy 3 Ifyou selected copy from the graph pane the following window will display click Copy If you selected copy from the lane pane skip to the next step Graph title Samples Metafile EMF E Bitmap PNG O Portable Document Format PDF 4 Open a document Word Excel PowerPoint etc Right click in the document and select Paste A graphic of the copied data view will be pasted into the document Copying Results Tables 1 Click in the peaks or capillaries pane 2 Select one or multiple rows 3 S
148. ate lids be used on sample plates Ifa lid is not detected a message will be displayed in the Start Run Wizard Compass software will reopen the sample tray to allow the user to insert a lid When inserting the sample plate ensure that the plate is firmly seated and level on the cold block Plates that are not level can interfere with the movement of the sample tray 7 Page 5 Data File The data file name will automatically default to the assay name appended with the current date and time To change the file name begin typing in the text box To change the directory where the data file will be stored click Browse Start Run Xs Begin the Automated Run The following protocol will be run You can change the results location and prefix Assay Protein Test Cycles 8 Schedule Overlapping with hold Run name 2012 10 16_09 29 06 Protein Test Browse Location C Users pfung Documents Compass Runs Click Start to begin the run Instrument status will change to running and the stop button and progress bar will display Compass Software for Peggy User Guide Starting a Run page 103 2012 05 30_ 15 52 06 DEX70K_2mil Running Stop LL Wed 3 58 PM Thu 11 28 AM a The run will continue until complete 14 19 hours depending on the assay Step 3 Post Run Procedures 1 Empty the capillary discard tray 2 Remove the assay plate 3 Dispose of the assay plate and capillar
149. bation reagent should be inserted then click Insert a 4th incubation step 4 icon in the Layout pane toolbar A new incubation reagent row will be added in the empty row or inserted above the selected row Compass Software for Peggy User Guide Creating a New Assay page 71 yEparatic Immobili Primary Seconda Tertiary Quaternz Detectior r a A fifth incubation reagent can now be added by repeating the above and clicking Insert a 5th incu bation step 5 icon instead NOTES When using the fourth and fifth incubation steps only five cycles can typically be performed per run as more water is needed per cycle for the additional wash steps Row J is the last row assignment that can be used on the assay plate New sample incubation or luminol rows cannot be inserted if row J already has an assigned reagent To insert a row in this case you must first move the contents of row J to another area on the plate To delete a row Click the row to be deleted then click Delete red x icon in the toolbar Only empty rows and fourth incubation rows can be deleted Rows for required assay reagents cannot be deleted xJOM 123 461 75 i i S B Cj 4 Dj 4 E F G H L J E Ze EO MMS ASB NOTE Samples antibodies and blocking buffer can be dispensed in Rows A J and in columns 1 12 or 13 24 Rows K P cannot be used for assay reagents Compass Software for Peggy User Guide page 72 Ch
150. blot can be customized To do this 1 Click the Edit Labels button The label box will display Compass Software for Peggy User Guide Changing the Virtual Blot View page 321 ke Graph H Image Lane OK Be he TO ol Ea eo Eg eo Tr rG if oe fs Eg a Lane Label F El Sample E Attribute se i CALE 2 Apt fl y Ss Aff og E Primary Ab E Attribute i a a oe a a an oe oe a a a g oy a gr Secondary Ab Attribute sol Capillary A TK EES a ee ee T N l N 4 Ir F 2 Check one or multiple label boxes and uncheck those you don t want to display To remove labels com pletely uncheck all boxes The following label options are available Sample Sample name If sample names were entered in the assay template Assay screen those names will display here Otherwise Sample default name will display Primary Primary antibody name If primary antibody names were entered in the assay template Assay screen those names will display here Otherwise Primary default name will display Secondary Ab Secondary antibody name If a secondary antibody name was entered in the assay template Assay screen those names will display here Otherwise Secondary default name will display Capillary Cycle and capillary number For example C1 3 indicates cycle 1 capillary 3 NOTE Peggy runs up to eight cycles 12 capillaries at a time so the cycle number displayed will alway
151. conds Exposure 2 60 seconds Exposure 120 seconds Exposure4 240 seconds Exposure 480 seconds Exposure6 960 seconds Peak Fit Analysis Settings page 231 3 Click OK to save changes and exit Sample data for the exposure selected will display in the Analysis screen Peak Fit Analysis Settings The peak fit analysis settings page lets you view and change peak fit settings for sample data To access these settings select Edit in the main menu and click Analysis then click Peak Fit in the options list NOTE Settings can be modified in an assay prior to starting a run or in a run file once the run has finished executing Analysis changes made to an executing run will not be saved to the final run file Analysis Simple Western ERK type filter text Peak Fit Advanced Images Analysis Settings Analysis Settings Peak Fit Peak Fit Peak Names Standards Minimum Range Maximum Baseline Threshold a Window Override Peak Find Apply To Threshold Width Click Import to import an analysis settings file This will be explained in more detail in Importing Analysis Settings on page 261 Compass Software for Peggy User Guide page 232 Chapter 8 Size Assay Data Analysis Click Export to export the current analysis settings file This will be explained in more detail in Exporting Analysis Settings on page 261 Click Restore Original to restore Compass default settings Click OK to save
152. culate sample protein molecular weight instead of the ladder The reported molecular weight for immunodetected sample proteins in Compass may vary slightly from predicted molecular weights based on sample and assay conditions To change the ladder capillary 1 Select Edit in the main menu and click Analysis then click Standards in the options list 2 Click the arrow in the drop down list next to Ladder Capillary then click a capillary number or none from the list Ladder Capillary none 1 MIW Positio 3 40 50 66 60 90 707 116 go 9 180 050 11 12 Add Remove Compass will use the data in the selected capillary to recalculate molecular weights for sample proteins in the run data using the information in the ladder table If none is selected Compass will instead use the information in the fluorescent peaks table fluorescent standards to calculate molecular weight for sample proteins NOTE When the ladder capillary is set to none the ladder table becomes inactive and cannot be modi fied Compass Software for Peggy User Guide page 252 Chapter 8 Size Assay Data Analysis Creating a New Standards Group 1 Select Edit in the main menu and click Analysis then click Standards in the options list 2 Click Add under the analysis settings box A new group will be created Analysis Settings Standards Standards 2 3 Click on the new group and enter a new name Analysis Settings Standards New St
153. cuments aall lt m File name Simple Western analysis X Save as type Analysis Settings settings X Hide Folder 4 The default directory is Compass Assays Change the directory if needed 5 Enter a file name and click Save The settings will be saved as a settings file Compass Software for Peggy User Guide page 263 Chapter 9 Charge Assay Data Analysis Chapter Overview Analysis Screen Overview Opening Run Files How Run Data is Displayed in the Analysis Screen Viewing Run Data Compass Run Data Notifications and Warnings Checking Your Results Group Statistics Copying Data Views and Results Tables Exporting Run Files Changing Sample Protein Identification Changing the Virtual Blot View Changing the Electropherogram View Closing Run Files Compass Analysis Settings Overview Advanced Analysis Settings Images Analysis Settings Peak Fit Analysis Settings Peak Names Settings Standards Settings Importing and Exporting Analysis Settings Compass Software for Peggy User Guide page 264 Chapter 9 Charge Assay Data Analysis Analysis Screen Overview The Analysis screen is used to view run data including electropherograms capillary images lane view data and tabulated results Any post run analysis is also done in this screen To access this screen click Analysis in the screen tab E assay C Run Summary Analysis Screen Panes The Analysis screen has six panes each displays the following data for up
154. d a E E a D 88822 888 Chapter 9 Charge Assay Data Analysis DeB pErk1 Erk ERK2 ppERK High phospho HeLa in 5 8 Ct 1 High phospho HeLa in 5 8 C1 3 High phospho HeLa in 5 8 Cis 62 63 EA ES EE E7 6A EJ Peak Names Checking this box will display peak name labels on all named peaks in the electrophe rogram NOTE If more than one peak label option is selected peak name labels will always be used for named peaks Compass Software for Peggy User Guide Changing the Electropherogram View page 333 Low phospho HeLa C12 1 400 300 200 100 000 200 200 700 600 500 400 300 200 a TI T TI wo a E E T a o 8 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 6B 69 70 pl Peak Values Checking this box will display the pl labels on all peaks in the electrooherogram NOTES When viewing standards and registration data this option is called Peak Positions Labels displayed are peak positions rather than pl If more than one peak label option is selected peak name labels will always be used for named peaks The reported pl for immunodetected sample proteins in Compass may vary slightly from predicted pls based on sample buffer and assay conditions Compass Software for Peggy User Guide page 334 Chapter 9 Charge Assay Data Analysis l Low phospho HeLa LT _ t Chemiluminescence 4004 3004 200 100 ooo 900 4 200 7
155. d click Filter 3 Check the Show Named Peaks only box and click OK eS Filter Capillaries SESE SSE SSS S82 S288 8 Show named peaks only Compass Software for Peggy User Guide Changing Sample Protein Identification page 189 Compass will hide peak data in the electropherogram and results tables for all unnamed peaks and instead only display data for named peaks in the electropherogram and results tables tel 2077n Samples SBS 8S8 2s Chemilumines cence So amp Capillaries Sample Primary Cap Peak Name MW kDa Height Area Area Width Compass Software for Peggy User Guide page 190 Chapter 8 Size Assay Data Analysis Changing the Virtual Blot View Options in the lane pane let users change the contrast or invert the virtual blot remove baseline noise change lane labels or overlay standards data on sample lanes The lane pane toolbar has the following options B Contrast Adjustment en Invert EF Edit Labels D Remove Baseline Overlay Standards Data Adjusting the Contrast 1 Click the Contrast Adjustment button The contrast tool will display IE Graph Image Lane Ra s aS gt gt gt F 2 gd amp gd gd a gt a gt eo NO eh E E OA De ONY G we YOK KO OKO OOO 180 2 Click the bar and drag it up or down to adjust the contrast 3 When finished click X to close the tool Compass Software for Peggy User Guide Changing the Virtual B
156. d eases 25 Step 1 Open a Template Assay 22060 eccndens 25 Step 2 Assign Assay Plate Reagents Optional 26 Step 3 Modifying the Assay Protocol Optional 30 Step 4 Add Assay Notes Optiondl 35 Step 5 Select a Schedule Optional J5 page ii Step 6 Add Assay Plate Annotations Optional 36 DFS aE TNO ASS OY rira a TAA 41 Step 8 Modify Default Analysis Parameters OAO nee ee eee ne E 42 Making Changes to an Existing ASSAY reen aiae 43 Switching Between Open ASSAYS 006 c cece eee e es 45 GREGHIIO G TEMDIGIC ASSOY oS x oii pi a Does 44 Viewing and Changing the Detection Exposures 45 COPVING PFOTOCOIS GING TCIM DHT OS era aaia 46 COPVINGIAN ASSAY FrOlO O haraa EREE 46 Copying an Assay Template meri eat 4 Pn oG TOOTO GNG TEMDIGTES rran EEE 47 PROUN GORAS FTO OC O wise EE 47 PHANG GUASSAY TEnest EAER 47 Importing and Exporting Protocols and Templates 48 Importing an Assay PrOlOCOl asirese iinn ait 48 Exporting an Assay Protool srusen 49 HN POTUAGONASSAY TENDI E i si vino tesetsavens 49 Exporting an Assay Template 06 5 50 Chapter 3 Running a Size Assay on Peggy gt SCH CAN EE AR EET E 52 O E a COG EE TEES 52 aaa ALLS a E T 52 Step 3 PO RON ProcedUTeS erercruevrriivereiy 59 DEO OWN U rE rT OEE 60 Chapter 4 Charge Assays aaa 61 AS AVSETT N aer eaa E E 62 AS OVS TEETE ATE ss tsi ches EAEE 62 Compass Software for Peggy User Guide S
157. d to detect peaks The mini mum value for this setting is 3 0 Larger widths help to eliminate the detection of shoulder peaks and noise peaks The default value is 15 0 Peak Fit Analysis Settings Groups Peak fit settings are saved as a group and multiple settings groups can be created Specific group settings can then be applied to individual capillaries sample names or attributes in the run data Compass Software for Peggy User Guide Peak Fit Analysis Settings page 233 NOTES ProteinSimple recommends using the Compass default values for peak fit analysis settings These settings are included in the default Peak Fit group Analysis settings are run file specific However settings can be imported or exported for use with other run files For more information see Importing and Exporting Analysis Settings on page 261 Peak fit groups are displayed in the analysis settings box Analysis Settings R ea n i p i The Peak Fit group shown contains the Compass default analysis settings Users can make changes to this group and create new groups To view settings for a group click on the group name in the analysis settings box Creating a New Peak Fit Group 1 Select Edit in the main menu and click Analysis then click Peak Fit in the options list 2 Click Add under the analysis settings box A new group will be created Analysis Settings Peak Fit Peak Fit 2 3 Click on the new group and enter a new name
158. de and should be saved Compass Software for Peggy User Guide Creating a New Assay page 35 Step 4 Add Assay Notes Optional 1 Click on the Notes tab 2 Clickin the pane and type any assay or protocol notes This information will be stored with the assay and run data Protocol Te Notes E This is our default assay for STATS analysis for samples run in triplicate NOTE When notes are being edited an asterisk will appear in the Notes tab to indicate changes have been made and should be saved Step 5 Select a Schedule Optional Peggy can execute cycles serially or in parallel To choose a schedule option select Edit and click Schedule 5 Serial O Overlapping Overlapping with hold Serial Each cycle is executed in sequential order and each cycle completes before the next one Starts Selecting this option result in the longest run times Overlapping Cycle steps are executed in parallel if timing permits shared use of the separation tray and robot Each cycle will run through to completion once started This option offers a reduction in overall run time without affecting the individual run time for each cycle Compass Software for Peggy User Guide page 36 Chapter 2 Size Assays Overlapping with Hold default Each cycle executes through the separate step in sequence and then enters a variable length hold step where capillaries remain in the incubation tray More sample stability is provided
159. dy to transfer the capillary box to the resource tray Place assay plate into the sample tray of the instrument and press Start Step 2 Start the Run ile You can start a run in one of two ways depending on what button is displayed in the status bar NEW RUN BUTTON click New Run Compass Software for Peggy User Guide Starting a Run a Select File in the main menu and click Open Assay rl Ready New Run Fi File Edit Instrument Window Help New Assay j Open Assay Protein Test Save Save As Peggy size Simple Western Demo Plate Import Protecal Simple Western Import Template Export Protocol Export Template f Separat l Separat Print j z gt Matrix f gt Blockin sr gt Primar page 53 b A list of the last five assays opened will display Select one of these assays or click Browse to open the Assay folder and select a different assay c Alternatively choose New Assay and select Peggy Size to get the default Peggy assay conditions d The Start button will display START BUTTON this indicates an assay is already loaded a Go to the Assay screen and verify this is the assay you want to use If not select File in the main menu click Open Assay and select another assay 2 Click Start This will launch the Start Run Wizard NOTE If the manifold was not cleaned prior to starting the run a message indicating this wi
160. e Graph Image Lane SK g he To Erkl pE 5 90 Area 13618 To view information for a band roll the mouse over a band until the info box appears NOTE The reported pl for immunodetected sample proteins in Compass may vary slightly trom predicted pls based on sample buffer and assay conditions Lane data displayed in the virtual blot is automatically aligned by Compass To view raw unaligned lane data and learn more about virtual blot viewing options see Changing the Virtual Blot View on page 319 Compass Software for Peggy User Guide How Run Data is Displayed in the Analysis Screen page 275 Peaks Pane Calculated Results Click the Peaks tab to view tabulated results for immunodetected sample proteins fluorescent standards or capillary registrations Each row in the table shows the individual results for each peak detected in each cap illary Data for samples in the peaks table is shown in the following example Cap Peak Name Position i Area ase High pho ERKL 2 ppERK2 711 2 62 4 ons 1538 7 NOTES Peaks that Compass names automatically using the user defined peak name analysis parameters are color coded The reported pl for immunodetected sample proteins in Compass may vary slightly from predicted pls based on sample buffer and assay conditions To view all rows Click the Peaks tab then use the scroll bar or click Maximize in the upper right corner Toresize columns Click the Peaks tab
161. e Graph View as an Image File BEOOR UF NOS aA TAA EXPONO RESU TAOIS riase AR Exporting Raw Sample Electropherogram Changing Sample Protein Identification Adding or Removing Sample Datd FIGING SOMDIC DONG E ETE E E Changing Peak Names for Sample Data Displaying Sample Data for Named Peaks CHGNGING TIC VINUG BIOUVICW srera ann POIUSUNO INC CONTOS aos a e TEE E ROTE VINTUGL BIO Suiunhe cctcsstanhacats aateuns PEE LEII AAE S CIS EEE E E Es whet Viewing the Uncorrected Sample Baseline Overlaying Standards Data on Sample Lanes Moving Lanes in the Virtual Blot View Changing the Electropherogram View Autoscaling the Electropherogram Compass Software for Peggy User Guide ee Stacking Multiple Electropherograms 198 Overlaying Multiple Electropherograms 199 ZOO eE TREA AEN TETOS 200 Customizing the Data Display 201 Selecting Data Viewing Options 210 Adding and Removing Baseline Points 216 Selecting the X Axis Molecular Weight Range 217 COST ONT E sah oot ata s NE EEEN 218 Compass Analysis Settings Overview 219 Advanced Analysis Settings c0 cece eeeee 221 Sarad OS eU GS os naclee Shame thts teeta 222 SAMPES OUNO sc cdi RA Hey eins wae 222 MAGE E O sa E EER IIN JD Advanced Analysis Settings Groups P Creating a New Analysis Group 223 Ch
162. e Sep Block 1 2 Detect Results F Fa a a 21G 12 54PM 12 56PM 2 00PM 2 16PM 5 26 PM 6 00 PM Assay Steps Charge based Assays Each assay step that is executed during the run is represented by an icon The time the individual step is scheduled to start is shown under each icon Details on what happens during each assay step is as follows sample ll 12 54 PM Description Sample Loading Step Capillaries are moved to the assay plate in the sample tray and samples are aspirated Capillaries are then transferred to the separation tray Separation Step Samples ampholyte mix and fluorescent pl standards are separated in the capillaries Capillaries are then exposed to UV light which immobilizes the separated proteins to the walls of each capillary During the separation voltage and current are mon itored and displayed in the IV Plot Separation of the fluorescent standards is recorded and a movie of the separation is compiled for viewing in the Separation pane after separation is complete Compass Software for Peggy User Guide page 114 Chapter 6 Run Status Description Primary Antibody 1 Step Capillaries are moved to the assay plate in the sample tray and primary antibody is aspirated Capillaries are then transferred to an incubator tray When incubation is complete Wash Buffer is aspirated Secondary Antibody 2 Step Capillaries are moved to the assay plate in the sample tray and secondary HRP c
163. e and primary antibody name so to use this feature this information must first be entered into the Assay template To do this a Click the Assay tab and go to the Template pane b Enter sample names and primary antibody names as described in Step 6 Add Assay Plate Annota tions Optional on page 79 Be sure to enter the same sample and or primary antibody names for the groups of samples you want to calculate statistics for In this example there are two sample types High phospho HeLa and Low phospho HeLa which were run with two different antibodies ERK 1 2 Primary 1 and ERK 1 2 Primary 2 Luminol and Peroxide Each of the two samples were run with the two antibodies in every cycle and the ERK1 2 antibody generates 6 named peaks 2 Select the Analysis tab and click the Grouping View icon in the view bar Clicking this icon toggles the grouping view on and off Compass Software for Peggy User Guide Group Statistics page 305 File Edit View Instrument Window Help u 00 3 Select a grouping option by clicking the arrow next to the grouping view icon These options allow you to group capillaries in multiple ways File Edit View Instrument Window Help east HlaHley E Cap Groups Group across cycles Grou p across runs Sample Primi Group across cycles Groups capillaries run in different cycles Group across runs Groups capillaries from multiple runs This option creates fewer groups and ge
164. e open files at the same time Toclose the run file being viewed Select File from the main menu and click Close e Toclose all open run files Select File from the main menu and click Close All Compass Software for Peggy User Guide Chapter 7 Controlling Peggy Chapter Overview Instrument Control Self Test Viewing and Changing System Properties Viewing Log Files Peggy s Status Modes Compass Software for Peggy User Guide page 119 page 120 Chapter 7 Controlling Peggy Instrument Control The Instrument menu allows users to control Peggy Window Help Mew Run Open Trays Manual Clean Cleanup Self Test Leveling Runs Properties Disconnect NOTE Instrument menu options are active only when a computer with Compass software is connected directly to Peggy Starting a New Run To start a new run select Instrument in the main menu and click New Run Then follow the steps described in Step 2 Start the Run on page 52 for size assays or Step 2 Start the Run on page 96 for charge assays Opening Trays To open any of the five trays select Instrument and click Open Trays The tray control window will appear Main Trays Other Trays wth Resources Samples Open a tray by clicking on its button The button will become highlighted indicating the tray is open Compass Software for Peggy User Guide Instrument Control page 121 NOTE Only one tray can be o
165. e run data Deleting a Peak Fit Group 1 Select Edit in the main menu and click Analysis then click Peak Fit in the options list 2 Click on the group in the analysis settings box you want to delete and click Remove Analysis Settings Peak Fit STAT peak fit 3 Click OK to save changes Applying Peak Fit Groups to Specific Run Data 1 Select Edit in the main menu and click Analysis then click Peak Fit in the options list 2 Click on the group in the analysis settings box you want to apply to specific run data Compass Software for Peggy User Guide page 236 Chapter 8 Size Assay Data Analysis Analysis Settings Peak Fit STAT peak fit 3 Application of peak fit groups to specific run data is done in the override box Click Add under the over ride box A default override data set will be created from sample information found in the run file Override Apply To Settings Biotinylated Ladder STAT peak fit Add Remove 4 Click the cell in the Apply To column then click the down arrow Override Apply To Settings tinylated Ladder STAT peak fit 5 Select an option from the drop down list This will apply the settings group selected to specific run data as follows Sample names All sample names entered in the assay template Assay screen will display in the drop down list otherwise the default name of Sample will be shown Select a sample name to apply group settings to all capillaries that use t
166. ea by the sum of all named peak areas for the capil lary and multiplying by 100 shown for named peak sample data only Width Displays the calculated peak width shown for sample data only S N Displays the calculated signal to noise ratio for the peak Capillaries Pane User Specified Peak Names Click the Capillaries tab to view tabulated results for sample proteins associated with specific primary anti bodies in the run data Compass labels these sample peaks automatically using user defined peak name set tings Each row in the table shows the individual results for the named peaks detected in each capillary Data for samples in the capillaries table is shown in the following example Primary Capillary ppErkl perl kl zza pERK2 High pho ERKL 2 Ci 1 Low phos ERKI 2 C1 High pho ERKL 2 C13 Low phos ERKL 2 ci High pho ERKL 2 Ci o OS ERKL 2 Cl NOTES Peaks that Compass names automatically with user defined peak name settings are color coded Information displayed for fluorescent standards and capillary registration data will be for identified stan dards or registration pedks To view all rows Click the Capillaries tab then use the scroll bar or click Maximize in the upper right corner Compass Software for Peggy User Guide How Run Data is Displayed in the Analysis Screen page 277 Toresize columns Click the Capillaries tab Roll the mouse over a column border until the sizing arrow appe
167. eak Name Columns An individual column per peak name will display for every peak identified by name in the run data Cells for capillaries in these columns will be blank if Compass did not find peaks automatically using the user defined peak name analysis parameters or none were entered To view area in the peak name columns Select in the upper right corner of the pane This displays the calculated percent area for the named peak compared to all named peaks This value results from dividing the individual peak area by the sum of all named peak areas for the capillary and multiplying by 100 Sample Primary Capillary Biotinylat Blocking C11 K562 anti E Chia K562 anti E C1 5 K562 anti E K562 anti E anti E f r r n Lam To view peak area in the peak name columns default Deselect in the upper right corner of the pane This displays calculated peak area for the individual peak only Compass Software for Peggy User Guide page 146 Chapter 8 Size Assay Data Analysis Viewing Run Data Each run file contains the following data for up to 96 capillaries Sample data For the immunodetected proteins in the sample Standards data For the fluorescent standards run with each sample Registration data For tracking capillaries as they are moved for various assay steps Data can be viewed for one all or individually selected capillaries Switching Between Sample Standards and Re
168. ect Edit in the main menu and click Analysis then click Advanced in the options list 2 Click the arrow in the drop down list next to Default then click a new default group from the list Analysis Settings Advanced STAT analysis Default Advanced Override Compass Software for Peggy User Guide page 354 Chapter 9 Charge Assay Data Analysis 3 Click OK to save changes Analysis settings in the group selected will be applied to the run data Modifying an Analysis Group 1 Select Edit in the main menu and click Analysis then click Advanced in the options list 2 Click on the group in the analysis settings box you want to modify Analysis Settings Advanced STAT analysis 3 Modify standards sample or image parameters as needed 4 Click OK to save changes The new analysis settings will be applied to the run data Deleting an Analysis Group 1 Select Edit in the main menu and click Analysis then click Advanced in the options list 2 Click on the group in the analysis settings box you want to delete and click Remove Analysis Settings Advanced STAT analysis 3 Click OK to save changes Applying Analysis Groups to Specific Run Data 1 Select Edit in the main menu and click Analysis then click Advanced in the options list 2 Click on the group in the analysis settings box you want to apply to specific run data Compass Software for Peggy User Guide Advanced Analysis Settings pa
169. ected sample proteins fluorescent standards or capillary registrations Data for samples is shown in the following example and immunodetected proteins are dis played as peaks kx Graph gt 23 Image Lane T Ar A Samples ppErk1 pErk1 Erk1 pERK2 ERK ppERK2 High phospho HeLa in 5 8 1 1 Chemilumines cence oSSRSRPSERBSZSSREBR SEE am ai sa Sa a aa ae is ey SE es Es ea ea eE E7 GE bg 0 Al pl More Graph view options will be described in more detail in Changing the Electropherogram View on page 325 Compass Software for Peggy User Guide How Run Data is Displayed in the Analysis Screen page 273 Image Pane Capillary Separation Image Data Click the Image tab to view final separation images of immunodetected sample proteins fluorescent stan dards or capillary registrations for up to 96 capillaries per experimental run Image data for samples is shown in the following example Graph w Image Lane Cycle 1 Samples Exp 240 0 W Compass Software for Peggy User Guide page 274 Chapter 9 Charge Assay Data Analysis Lane Pane Virtual Blot Like Image Data Click the Lane tab to view data for immunodetected sample proteins fluorescent standards or capillary reg istrations as bands in individual lanes This virtual blot like image is similar to traditional blot results Data for samples in the lane view is shown in the following example and immunodetected proteins are displayed as bands k
170. ed analysis settings These set tings are included in the default Advanced group Analysis settings are run file specific However settings can be imported or exported for use with other run files For more information see Importing and Exporting Analysis Settings on page 386 Analysis groups are displayed in the analysis settings box Analysis Settings Add Remove The Advanced group shown contains the Compass default analysis settings Users can make changes to this group and create new groups To view settings for a group click on the group name in the analysis settings box Creating a New Analysis Group 1 Select Edit in the main menu and click Analysis then click Advanced in the options list 2 Click Add under the analysis settings box A new group will be created Analysis Settings Advanced Advanced 2 s 3 Click on the new group and enter a new name Compass Software for Peggy User Guide Advanced Analysis Settings page 353 Analysis Settings 4 Modify standards sample or image parameters as needed 5 To use the new group as the default analysis settings for the run file data click the arrow in the drop down list next to Default then click the new group from the list Analysis settings in the new group will then be applied to the run data Analysis Settings Advanced STAT analysis Default Override 6 Click OK to save changes Changing the Default Analysis Group 1 Sel
171. elect Edit in the main menu and click Copy or right click and select Copy A Open a document Word Excel PowerPoint etc Right click in the document and select Paste Data for the rows selected will be pasted into the document Saving the Graph View as an Image File 1 Click in the graph pane 2 Select Edit in the main menu and click Copy or right click and select Copy 3 Select an image option EMF PNG or PDF in the pop up window then click Save Compass Software for Peggy User Guide Exporting Run Files page 311 Graph title Samples O Metafile EMF Bitmap PNG E Portable Document Format PDF 4 Select a directory to save the file to and enter a file name then click OK Exporting Run Files Results tables and raw plot data can be exported for use in other applications Exporting Results Tables To export the information in the peaks and capillaries tables 1 Click File in the main menu and click Export Tables 2 Selecta directory to save the files to and click OK Data will be exported in txt format NOTE To exclude export of standards data or export results table data in csv format see Setting Data Export Options on page 391 Exporting Raw Sample Electropherogram Data To export raw sample plot data 1 Click File in the main menu and click Export Spectra Compass Software for Peggy User Guide page 312 Chapter 9 Charge Assay Data Analysis Edit View Instrument Window Help
172. ents Compass Assos tf Sexxrasoys P on Organize v New folder Fr Favorites Documents library MB Desktop Assays B Downloads Name E Recent Places Arrange by Folder Date modified Type SW long incubation assay 9 29 2011 3 27 PM Compass Assay File Z Simple Western 2 assay 9 29 2011 3 24 PM Compass Assay File Lib J Test assay assay 9 29 2011 3 21 PM Compass Assay File raries gt Simple Western assay 9 29 2011 3 18 PM Compass Assay File E i Erk Assay assay 9 12 2011 1 11 PM Compass Assay File Simon Decontamination Procedure assay 9 12 2011 1 11 PM Compass Assay File Generic Simple Western 9 9 2011 assay 9 9 2011 7 18 PM Compass Assay File Simple Western Demo Plate assay 9 9 2011 7 18 PM Compass Assay File File name My New Assay m S Hide Folders E NOTE New assays are saved in the Compass Assays directory Compass Software for Peggy User Guide page 42 Chapter 2 Size Assays Step 8 Modify Default Analysis Parameters Optional You can preset the parameters used to analyze run data generated with the assay 1 Select Edit from the main menu and click Default Analysis The following screen will display Analysis Settings Analysis Settings Advanced Advanced Standards Peak Width Allowable Drift Sample Peak Fit Starting Width Ratio ow Image ona ned y Adva Median Filter Theechold Ratio Override Median Filter Threshold Limit Restore Original 2 Pro
173. er Guide Changing Sample Protein Identification page 185 Hiding Sample Data You can hide the results for a sample protein in the results tables without completely removing it from the reported results To to this i 2 Click Show Samples in the View bar Click Single View in the View bar Click on the row in the experiment pane that contains the sample you wish to correct then click the Graph tab Right click the peak in the electropherogram or peaks table and select Hide peak Compass will hide the peak data in the results tables al oon ae peas T Br To A st Chemilumines cence BeRSERE BEES Zoom Out z Remove Peak Hide X Name Add Peak Add Baseline Point o S MW kDa HE Peaks gt EH Capillaries Remove Baseline Point Sample Primary Cap Peak Name Position MW kDa Height Clear All E E E R E Copy cutee Compass Software for Peggy User Guide page 186 Chapter 8 Size Assay Data Analysis 5 To view hidden peak data click View in the main menu and click Show Hidden Hidden peak data will display in the results table and be marked with an X Chemilumines cence 40 66 30 116 MW kDa HH Peaks gt EH Capillaries 0O Sample Primary Cap Peak Name Position MW kDa Height Area 9 Area Width anti E 6 Tounhide a peak right click on the peak in the electropherogram or peaks table and select Unhide Peak Changing Peak Names fo
174. er to change The following screen displays Change user ProteinSim x W 7 e gt C 4 10 1 2 18 8000 admin auth user 10 Welcome admin Change password Log out Home gt Auth gt Users gt steveg Change user Username steveg Required 30 characters or fewer Letters digits and _ only algorithm pbkdf2_sha256 iterations 10000 salt bO83Ho hash Password Wah ce bg EEE ee ee ee ee ee ee oe ee eee ee ee oe ie ee oe not stored so there is no way to see this user s password but you can change the password First name Last name Gallagher Email address Active Designates whether this user should be treated as active Unselect this instead of deleting accounts _ Staff status Designates whether the user can log into this admin site 2 Raw passwords are not stored they must be changed manually Click the text link to access the pass word change form Compass Software for Peggy User Guide Authorization Server Change password steveo XW J gt 10 1 2 18 3000 admin auth user 10 password Home Auth gt Users steveg Change password Change password steveg Enter a new password for the user steveg Password again Enter the same password as above for verification 3 Enter the new password then click Change password Encryption Details page 419 Welcome admin Change password Log out Change password Compass uses the SHA1 hash algor
175. ettings file This will be explained in more detail in Exporting Analysis Settings on page 387 Click Restore Original to restore Compass default settings Click OK to save changes and exit Click Cancel to exit without saving changes Standards Settings Peak Width The approximate width at full width half max used to filter out fluorescence artifacts which improves recognition of standards The default value is 15 Allowable Drift The distance in pixels that standards are expected to move compared to their entered positions in the standards analysis page This setting assists in recognition of standards The default value is 100 Sample Settings Peak Fit Starting Width Ratio Focuses the peak fit towards the peak center and aids the overall peak fitting The default value is 0 5 Image Settings Median Filter Threshold Ratio Pixel ratio used to filter out camera artifacts The default value is 0 5 Median Filter Threshold Limit Pixel threshold value used to filter out camera artifacts The default value is 100 Compass Software for Peggy User Guide page 352 Chapter 9 Charge Assay Data Analysis Advanced Analysis Settings Groups Advanced analysis settings are saved as a group and multiple settings groups can be created Specific group settings can then be applied to individual capillaries sample names or attributes in the run data NOTES ProteinSimple recommends using the Compass default values for advanc
176. for size assays or page 278 for charge assays and Using Groups on page 175 for size assays or page 304 for charge assays u tE afl i Compass Status Bar The status bar is located in the lower right corner of the main window It displays active software processes and their progress Analyzing Simple Western ERK Demo E Software Menus A brief description of the software menus in the main menu are described in this section Not all menus are available in every screen and menu commands change depending on what screen is active The menus and commands available for each screen will be detailed in the individual screen sections Compass Software for Peggy User Guide page 8 Chapter 1 Getting Started with Compass File Menu The File menu contains basic file commands File Edit View Instrument Open Run Add Run Close Close All Save Save As Export Tables Export Spectra Exit Edit Menu The Edit menu contains basic editing commands analysis and preferences options Preferences is described in Chapter 10 Setting Custom User Preferences Edit Instrument Window Help Cut Copy Ctrl C Paste Ctrl V Default Analysis Analysis Preferences View Menu The View menu is only available in the Analysis screen and allows users to change how data is displayed Instrument Window Single View Multiple View Standards Registration Samples Filter View Region S
177. ftware for Peggy User Guide Run Summary Screen Overview page 107 amp 2012 03 05_11 51 19_HelaControlERKassay Compass Co let File Edit Instrument Window Help E Assay ak Analysis Run 2012 03 05_11 51 19_HelaControlERKassay E Separation lt IV Plot z YY Status gt Run 2012 03 05_11 51 19_HelaControlERKassay Path CAUsers pfung Documents ProteinSimple 2012 03 05 11 51 19 HelaControlERKassay cl Assay HelaControlERKassay Schedule Overlapping with hold Instrument PLOOO4 PLOO04 Started Mon 11 56 AM Mar 5 2012 PST Completed Tue 6 35 AM Mar 6 2012 PST Cycle Sample Sep Hold 1 2 J Detect Results 2 ray et 3 0 E SS 1 LN t mmr 11 56AM 12 01PM 1 10PM 6 10PM 6 36PM 8 45PM 953PM 10 27PM TY 1 Dy 2 3 T po 2 Bo n M B a o 1 10PM 1 14PM 2 24PM 7 27PM 7 53PM 10 02PM 11 10PM 11 44 PM TY ry r 3T m GER 3 a mes ot 2 24PM 2 28PM 3 37PM 8 46PM 9 13PM 11 22PM 12 30AM 1 04AM TT rer P 3 T Corl 2 4 tee ll aen Soir 3 37PM 3 42PM 4 51PM 10 04PM 10 30PM 12 39AM 1 47AM 2 21AM Ty War PT 3 T Gl ee 5 Wo t cee gt lt I gt B 4 51PM 4 56PM 6 05PM 10 58PM 11 24PM 1 33AM 242AM 3 15AM mT 67 PT 3T m r r 6 HH L Simerjeet 6 05 PM 6 09 PM 7 18PM 11 39PM 12 05AM 214AM 3 22AM 3 56AM TN a gt pyan zT m Ba 7 LL G ae 7 19 PM 7 23 PM 8 32PM 12 20AM 12 46AM 2 55AM 4 03AM 4 37AM CTY War zT 3 T Gal ss 8 LN be 8 32
178. ge 355 Analysis Settings Advanced 3 Application of analysis groups to specific run data is done in the override box Click Add under the over ride box A default override data set will be created from sample information found in the run file Override Apply To Settings Hi phospho HeLa HeLa 4 Click the cell in the Apply To column then click the down arrow Override Apply To Settings i phospho HeLa HeLa Hi phospho HeLa Low phosphe HeLa Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle amp Cycle 7 Cycle 8 Custom Settings 5 Select an option from the drop down list This will apply the settings group selected to specific run data as follows Sample names All sample names entered in the assay template Assay screen will display in the drop down list otherwise the default name of Sample will be shown Select a sample name to apply group settings to all capillaries that use this sample name in the run file Attributes All sample attributes entered in the assay template Assay screen will display in the drop down list Select an attribute to apply group settings to all capillaries that use this attribute in the run file Compass Software for Peggy User Guide page 356 Chapter 9 Charge Assay Data Analysis Cycle 1 8 When this option is selected group settings will be applied to all capillaries in the cycle Custom settings Lets you choose specific capillaries to apply the group settings t
179. ggy User Guide page 298 Chapter 9 Charge Assay Data Analysis To set an unidentified peak as a standard Right click the peak in the electropherogram or peaks table and select Force Standard Compass will assign the peak as a standard and cor rectly reassign the remaining standard peaks A lock icon indicating the standard was set man ually will display next to the peak in the peaks table NOTE To remove standards peak assignments that were made manually right click on the peak in the electropherogram or peaks table and click Clear c Repeat the previous steps for the remaining rows in the experiment pane to make sure all standards are identified correctly Lane Pane a Click Multiple View in the View bar b Click on the first row in the experiment pane then click the Lane tab Standards will be bands and identified with a green outline Check that each lane has the appropriate number of fluorescent pl standard bands for the pl Standard Ladder you are using They will also be identified with a green S in the peaks table The pl standard bands at the low and high end of the pl range in each lane will display greater intensity as they are at higher concentrations and are also used as registration stan dards In the following example the pl standards shown are those for pl Standard Ladder 3 P N 040 646 To view band labels roll the mouse over the individual bands Compass Software for Peggy User Guide Checking Your Results page
180. ght in kDa for the sample protein Compass Software for Peggy User Guide Peak Names Settings page 241 Analysis Settings ERK1 2 Name MW Color Range Show eR 42 D 8 Click in the first cell in the Color column then click the button Analysis Settings ERKL 2 Name MW Color Range 7 Show ERK2 42 o oity 10 The color selection box will display BEEBE EE Bee EE See EE eee l l 1 l E E C oTr E E A stom colors EE HHEHEHHMTI Define Custom Colors gt gt The color selected will be used to identify the sample protein peak in the peaks and capillaries table in the Analysis screen 9 Click a color or define a custom color and click OK The color selection will update in the table Analysis Settings EREL 2 Mame MW Color Range Show ERK2 42 yy 10 10 Click in the first cell in the Range column Compass Software for Peggy User Guide page 242 Chapter 8 Size Assay Data Analysis Analysis Settings ERKL 2 Mame MW Color Range Show ERK2 M Eo 11 Enter a range window for the MW entered Compass will automatically name peaks found within this percent of the molecular weight For example if the molecular weight entered is 40 kDa and a 10 range is used all peaks between 36 and 44 kDa will be identified with this peak name NOTE The reported molecular weight for immunodetected sample proteins in Compass may vary slightly from p
181. gistration Data Views You can switch between viewing sample standards and registration data in a run file using the View bar or View menu B Instrument Window Single View Multiple View Standards Registration Samples Filter View Region Show Hidden Data buttons in the View bar Show Standards Show Registrations Show Samples Compass Software for Peggy User Guide Viewing Run Data page 147 To view sample data Click Show Samples in the View bar or select View in the main menu and click Samples 7 File Edit View Instrument Window Help Assay CF Run Summary EE Analysis J s pura rE S a Sample biotin hela hela hela hela hela hela hela hela hela hela biotin Blocki hela hela hela hela hela hela hela hela hela hela hela biotin hela hela hela hela hela hela hela hela hela hela hela biotin hela hela hela hela Primary Cyc Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2
182. gistration Force Compass will assign the new peak as the cap illary registration A lock icon indicating the registration was set manually will display next to the peak in the peaks table NOTE To remove registration peak assignments that were made manually right click on the peak in the electropherogram or peaks table and click Registration Clear Repeat the previous steps for the remaining rows in the experiment pane to make sure all registrations are identified correctly Step 4 Checking the Ladder The biotinylated ladder should have six sizing standards at 12 40 66 90 116 and 180 kDa To verify the lad der standards are identified correctly 1 2 Click the Analysis screen tab Click Show Samples in the View bar Verification that the ladder standards have been correctly identi fied can be done in either the graph or lane panes but manual corrections must be done in the graph pane Graph Pane a Click Single View in the View bar b Click on the row in the experiment pane that contains the ladder typically row 1 then click the Graph tab Check that the electrooherogram has six ladder peaks labeled Ldr 12 Ldr 40 Ldr 66 Ldr 90 Ldr 116 and Ldr 180 Compass Software for Peggy User Guide page 170 Chapter 8 Size Assay Data Analysis 2012 03 05 11 51 19 HelaControlERKassay Co File Edit View Instrument Window Help E as g7 E Assay 1g Run Summary m G amp Graph a Image Lane 7
183. gs Groups Advanced analysis settings are saved as a group and multiple settings groups can be created Specific group settings can then be applied to individual capillaries sample names or attributes in the run data NOTES ProteinSimple recommends using the Compass default values for advanced analysis settings These set tings are included in the default Advanced group Analysis settings are run file specific However settings can be imported or exported for use with other run files For more information see Importing and Exporting Analysis Settings on page 261 Analysis groups are displayed in the analysis settings box Analysis Settings Add Remove The Advanced group shown contains the Compass default analysis settings Users can make changes to this group and create new groups To view settings for a group click on the group name in the analysis settings box Creating a New Analysis Group 1 Select Edit in the main menu and click Analysis then click Advanced in the options list 2 Click Add under the analysis settings box A new group will be created Analysis Settings Advanced Advanced 2 Add Remove 3 Click on the new group and enter a new name Compass Software for Peggy User Guide page 224 Chapter 8 Size Assay Data Analysis Analysis Settings Advanced 4 Modify standards sample or image parameters as needed 5 To use the new group as the default analysis settings for the run
184. guides with a folder let you know that if the pane is dropped at that location it will be added as a new tab in an area with one or more pane tabs Area guides with a window let you know that if the pane is dropped at that location it will become a separate window outside the Compass main window Compass Software for Peggy User Guide Changing the Compass Main Window Layout page 13 The following figure shows the Analysis screen after moving the Graph pane File Edit View Instrument Window Help EEE Assay CH Run summary FAE EH Experiment E Graph gt BH Image E Lane F g B 7 oE peaks _ EE Capillaries m a Sample Primary C Sam ples Sample Primary Cap Peak Name Position MW kDa Height Area Area Width S N E biotin Blocki 1 g biotin lad Blocki fen 1 Ldr12 413 12 522 8 12879 231 871 hela Blocki 1 c v biotin lad Blocki fon 2 Ldr40 629 40 699 7 16404 220 121 2 hela Blocki 1 8 1500 Ldr 116 biotin lad Blocki fon 3 Ldr66 739 66 682 5 13319 183 1474 hela Blocki 1 D Ldr 40 a Y biotin lad Blocki 4 Ldr90 716 90 1598 4 29341 17 2 ia hela Blocki 1 Ei J Ldr 6g Ldr 80 biotin lad Blocki 5 Ldr116 812 116 1039 1 21661 19 6 E v Y hi in ladder hela Blocki 1 5 biotin lad 6 ae eL a L hela Blocki 1 o hela Blocki 1 8 0 hela Blocki 1 3 1131 6 n 56 7 e hea Block 1 8 1501 ERR e
185. gy User Guide page 86 Chapter 4 Charge Assays Making Changes to an Existing Assay 1 Select File in the main menu and click Open Assay Edit Instrument Window Help New Assay i Open Assay j WIEK assay Protein Test Save oe Peggy Charge Peggy Charge 1017 Import Protecol Erk Assay Import Template Browse Export Protocol Export Template Print a rono p Exit 2 Alist of the last assays opened will display Select one of these assays or click Browse to open the assay folder and select a different assay 3 Follow the steps in Creating a New Assay on page 67 to make changes and save the assay Switching Between Open Assays If more than one run file is open you can switch between viewing the assays for each run To do this 1 Click the down arrow in the run box File Edit Instrument Window Help Run HeLa IKb test ka HUT78 Screen Lay one 2 Select the run for the assay you want to view from the drop down list Compass Software for Peggy User Guide Creating a Template Assay page 87 Creating a Template Assay Users can create and save template assays that can be used as a starting point for creating new assays To do this 1 Select File in the main menu Click Open Assay to open an existing assay or click New Assay to open an existing template assay 2 Follow the steps in Creating a New Assay on page 67 to make changes to the assay 3 When changes
186. h Soak Time sec 4 Detection Well Row Wash Load Time sec Detection Profile Cyclel Al 25 0 Power 1 Step 3 0 100 0 2 20 0 150 0 120 0 2 0 2 20 0 150 0 60 0 qa 2 0 2 20 0 150 0 10 0 ae 2 0 2 20 0 150 0 F1 2 0 5 Exposures page 73 2 Five incubation steps are allowed per protocol Users can select the type of incubation for each step The available incubation types and their default Simple Western use is as follows To change the type click the incubation step name and select an option from the drop down list First incubation Primary antibody Second incubation Secondary antibody Third incubation User defined tertiary antibody Fourth incubation User defined quaternary antibody Fifth incubation User defined custom Primary Ab Quaternary Ab Custom 3 If needed change the primary incubation time To do this click the cell in the value column next to Pri mary Ab Time min and enter a new value in minutes Compass Software for Peggy User Guide page 74 Chapter 4 Charge Assays Add Removwe gt Sample t Separation b Immobilization Time sec 100 0 4 Primary Ab Time min 40 0 Well Row ee Load Time sec 2 0 Washes 2 Wash Load Time sec 20 0 Wash Soak Time sec 150 0 Secondary Ab Time min 60 0 b Tertiary Ab Time min 10 0 Detection 4 If needed change the primary incubation reagent row location To do this c
187. he cell in the Apply To column then click the down arrow Override Apply To Settings i phospho HeLa STAT peak fit Low phosphe HeLa 1 mg mL Cyclel Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycles Custom Settings 5 Select an option from the drop down list This will apply the settings group selected to specific run data as follows Sample names All sample names entered in the assay template Assay screen will display in the drop down list otherwise the default name of Sample will be shown Select a sample name to apply group settings to all capillaries that use this sample name in the run file Attributes All sample attributes entered in the assay template Assay screen will display in the drop down list Select an attribute to apply group settings to all capillaries that use this attribute in the run file Cycle 1 8 When this option is selected group settings will be applied to all capillaries in the cycle Custom settings Lets you choose specific capillaries to apply the group settings to When this option is selected the following pop up box appears to let you enter a specific capillary number or range of capillaries Compass Software for Peggy User Guide page 366 Chapter 9 Charge Assay Data Analysis Enter cycle and capillary descriptor Examples 3 1 1 12 Cycle 2 capillary 3 and cycle 1 capillaries 1 through 12 1 3 6 8 10 Cycles 1 through 3 capillaries 6 8 and 1
188. he icon displays warning details Sample Primary 5 2 Sample Primar 5 3 Standards Warn ing Low Confidence Manual correction of standards data notification Indicates the user modified the standards data manually Rolling the mouse over the icon displays the type of data modification made Qo Registrations warning Indicates that a capillary registration was not identified prop erly This can be resolved by manually identifying the registration peak Please refer to Step 3 Checking Capillary Registrations on page 168 for details Rolling the mouse over the icon displays warning details Compass Software for Peggy User Guide Checking Your Results page 163 e Manual correction of registrations notification Indicates the user modified the capillary registration data manually Rolling the mouse over the icon displays the type of data modification made Peak fit warning Indicates that a peak cannot be fit properly This is typically caused either by a very small peak in the electrooherogram or a peak that is very close to the end of the molecular weight range This can be resolved by removing the peak Please refer to Step 4 Checking the Ladder on page 169 or Step 5 Checking Samples on page 171 for details Rolling the mouse over the icon displays warning details 4s Kitlow pho anti H 2 4 Kitlow pho anti B 2 5 Peak Fit Warning Too many iterations f Checking Yo
189. hemiluminescence ppErk1 ato Plog RK aaa SE 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 pl E Capillaries Sample Primary Cap Peak Name Position p Height Area Area Width Low p ERKL 2 C6 33 00361 Compass Software for Peggy User Guide Changing the Virtual Blot View Changing the Virtual Blot View page 319 Options in the lane pane let users change the contrast or invert the virtual blot remove baseline noise change lane labels or overlay standards data on sample lanes The lane pane toolbar has the following options B Contrast Adjustment ee Invert Edit Labels D Remove Baseline Overlay Standards Data Adjusting the Contrast 1 Click the Contrast Adjustment button The contrast tool will display og Graph Image Lane POPEL EPELE of OPAL POOR A Ae a PP EP EIEIO OS R 5 Seek eke eer Soe PPS 2 Click the bar and drag it up or down to adjust the contrast 3 When finished click X to close the tool Compass Software for Peggy User Guide Sti Dba 7o page 320 Chapter 9 Charge Assay Data Analysis Inverting the Virtual Blot 1 Click the Invert button The virtual blot image will invert hire Graph 1E Image Lane PEPLE PEEL PEPEE EE i oe Pe a oe oP al oF oP a8 oh ok ok oP ot oh ar ae ot ot wn 2 Click the Invert button again to return to the default view Selecting Lane Labels The labels shown above the lanes in the virtual
190. his sample name in the run file Compass Software for Peggy User Guide Peak Fit Analysis Settings page 237 Attributes All sample attributes entered in the assay template Assay screen will display in the drop down list Select an attribute to apply group settings to all capillaries that use this attribute in the run file Cycle 1 8 When this option is selected group settings will be applied to all capillaries in the cycle Custom settings Lets you choose specific capillaries to apply the group settings to When this option is selected the following pop up box appears to let you enter a specific capillary number or range of capillaries woven Enter cycle and capillary descriptor Examples 2 3 1 1 12 Cycle 2 capillary 3 and cycle 1 capillaries 1 through 12 1 3 6 8 10 Cycles 1 through 3 capillaries 6 amp and 10 Cancel 6 Ifyou need to change the peak fit group used for a data set click the cell in the Settings column and click the down arrow Select a group from the drop down list Override Apply To Settings a STAT peak fit 7 Repeat the previous steps to apply other groups to specific run data 8 To remove a data set click on its cell in the Apply To column then click Remove 9 Click OK to save changes Compass Software for Peggy User Guide page 238 Chapter 8 Size Assay Data Analysis Peak Names Settings The peak names analysis settings page lets you enter custom naming set
191. how Hidden Compass Software for Peggy User Guide Software Menus page 9 Instrument Menu The Instrument menu is only available when Compass is connected to Peggy directly Instrument control options are explained in Chapter 7 Controlling Peggy Window Help New Run Open Trays Manual Clean Cleanup Self Test Leveling Runs Properties Disconnect Window Menu The Window menu allows users to switch between Assay Run Summary or Analysis screens and restore screens to the Compass default layout Help Assay Run Summary Analysis Default Layout Assay Displays the Assay screen which is used to create view and edit assays Run Summary Displays the Run Summary screen which is used to monitor the status of a run in progress Analysis Displays the Analysis screen which is used to view sample electrooherograms lane data and results Default Layout Restores the individual panes in the current screen back to their default size and location Compass Software for Peggy User Guide page 10 Chapter 1 Getting Started with Compass Help Menu The Help menu provides access to Help software updates release notes and other software information 7 Help Contents Check for Updates Release Notes Compass Log About Compass Help Contents Displays the Compass Help file Check for Updates Automatically checks to see if a new version of Compass is available Release Notes Dis
192. i hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki i hela Blocki hela Blocki i ERK2 k MW kDa 42 hela Blocki Area 44283 hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hala Rlarbi nD a NNN N N e RP RP RP RP BP RP BP BP Pe Sample ial Cap Peak Name Position MW kDa Height Area Area Width 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 A Compass Software for Peggy User Guide Group Statistics page 175 Step 6 Assigning Peak Names Optional Compass can identify and automatically name sample proteins associated with specific primary antibodies in the run data using user specified peak name settings For more information on how to do this see Peak Names Settings on page 238 Group Statistics The Grouping View is used to analyze replicates by calculating the mean standard deviation and CV of named proteins see Peak Names Settings on page 238 for detailed information on entering named pro teins Statist
193. i phospho HeLa Erk C7 Zoom Out Add Peak Add Baseline Point Remove Baseline Point Clear All BR ee sg Chemilumines cence g Copy 5650 5 675 5 700 5 725 5750 5 775 5800 5825 5850 5875 5900 5925 5950 5975 6 000 pl Customizing the Data Display Electrooherogram peak labels plot labels and display options can be customized by the user To do this select the Graph Options button Compass Software for Peggy User Guide Changing the Electropherogram View page 331 Ie Graph Image 1700 1600 ppErk1 pErk1 Erk1 pERK2 T2202 o 1500 ppERK Peak Names 1400 Peak Values 1300 1200 E ad Fitted Peaks a 1100 E J amp Baseline Fit 1000 Grid Lines 500 E Plot Label 5 0 W Sample C Attribute Le E Primary C Attribute D Capillary Exposure 500 400 300 200 100 0 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 pl Peak Labels You can customize the labels used to identify peaks in the electrooherogram with these options TT Matching Peak Names F Peak Names Peak Values Matching Peak Names Checking this box will draw vertical lines through each named peak Using this option with Stack the Plots or Overlay the Plots features is useful for visual comparison of named peaks across multiple capillaries Compass Software for Peggy User Guide page 332 ke en e Chemilumine 7 a o io a E a E a D a o a o fin
194. ick Clear All Selecting the X Axis pl Range The pl range used for the x axis can be changed To do this Select View in the main menu and click View Region The following pop up window will display g View Region E Full Range Lower 5 0 Upper e To change the x axis pl range displayed for the run data Enter new values in the Lower or Upper range in pl and click OK Electropherogram and virtual blot data will update to display only the data in the entered range Compass Software for Peggy User Guide Closing Run Files page 347 e Tosee the full x axis pl range included in the run data Check Full Range Electropherogram and virtual blot data will update to display the full range of available data lk Graph gt H Image Lane EB E Samples 1100 ppErk1 Erk ERK2 pErki pERK2 ppERK2 z Hi phospho HeLa Ctl Chemilumines cence BeBe EE SS o 425 450 475 500 525 550 575 GOD 625 650 675 700 725 750 775 800 825 850 875 900 925 pl NOTE You can change the default x axis range that Compass uses For more information see Peak Fit Analysis Settings on page 360 Closing Run Files If more than one run file is open you can close just one file or all the open files at the same time Toclose one of multiple open run files In the experiment pane click on one of the sample rows in the file Next click File from the main menu and click Close e To close all open run f
195. ics for each protein are also plotted for easy comparison between samples antibodies and pro teins Using Groups 1 A group is automatically created for capillaries with the same sample and primary antibody name so to use this feature this information must first be entered into the Assay template To do this a Click the Assay tab and go to the Template pane b Enter sample names and primary antibody names as described in Step 6 Add Assay Plate Annota tions Optional on page 36 Be sure to enter the same sample and or primary antibody names for the groups of samples you want to calculate statistics for In this example there are two sample types Sample A and Sample B which were run with two dif ferent antibodies Primary 1 and Primary 2 Compass Software for Peggy User Guide page 176 Chapter 8 Size Assay Data Analysis K Detection Each of the two samples were run with each of the two antibodies twice in every cycle This creates four groups for the combination of two samples and two antibodies 2 Select the Analysis tab and click the Grouping View icon in the view bar Clicking this icon toggles the grouping view on and off File Edit View Instrument Window Help 3 Select a grouping option by clicking the arrow next to the grouping view icon These options allow you to group capillaries in multiple ways File Edit View Instrument Window Help eet af ks E Cap Groups i Sample Pri
196. ied as a ladder peak Right click the peak in the electrophero gram or peaks table and select Remove peak Compass should correctly reassign the remain ing peaks as ladder standards To set an unidentified peak as a ladder peak Right click the peak in the electrooherogram or peaks table and select Add Peak Compass will assign the peak as a ladder standard and correctly reassign the remaining ladder standards peaks NOTE To remove ladder peak assignments that were made manually and go back to the original view of the data right click in the electropherogram and click Clear All Compass Software for Peggy User Guide Checking Your Results page 171 Lane Pane a Click either Single View or Multiple View in the View bar b Click on the row in the experiment pane that contains the ladder typically row 1 then click the Lane tab Check that the lane has six ladder bands labeled Ldr 12 Ldr 40 Ldr 66 Ldr 90 Ldr 116 and Ldr 180 0 To view band labels roll the cursor over the individual bands If ladder bands are not iden tified correctly they must be corrected in the graph pane as described in the previous section pared iia k gt a a ee amp ES st fg E Assay Pg Run Summary EE Analysis E Experiment D IE Graph 2 Image E Lanen H s o Primary Cycle ss biotin Blocki 1 Ka Ka hein Block 1 Il yng SP PPP PILI IP IIE SIP PPO P PPO PPS OME
197. ies Disposal will depend on the samples that have been assayed If sample origins are unknown ProteinSimple recommends that used capillaries and plates be disposed of in biohazard waste WARNING SHARPS HAZARD The capillaries may present a potential sharps hazard Dispose of used capillaries in biomedical waste sharps containers IWARNING BIOHAZARD A Samples and waste bottle contents should be handled by procedures recommended in the CDC NIH manual Biosafety in Microbiological and Biomedical Laboratories BMBL The manual is available from the U S Government Printing Office or online at http www cdc gov biosafety publications bmbl5 Depending on the samples used waste bottle contents may constitute a biohazard Use precaution when emptying the waste bottle Dispose of waste bottle contents in accor dance with good laboratory practices and local state provincial or national environmen tal and health regulations Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste vial before you store han dle or dispose of chemical waste Compass Software for Peggy User Guide page 104 Chapter 5 Running a Charge Assay on Peggy Stopping a Run 1 To stop a run click Stop When the run stops instrument status will go to Not Ready and a Cleanup but ton displays a NOTE Ifa run is stopped prior to completion Peggy must perform a cleaning protocol to remove used Not
198. ile is shown in the Status pane vant r Al Path CAUsersiptung Documents Proteinsimpleye01 2 03 05 11 51 14 HelaControlERKassay c Assay HelaControlERRassay schedule Overlapping with hold Instrument FPLOOO4 PLOOO4 started Mon11 56 AM Mar 5 2012 PST Completed Tue 6 35 AM Mar 6 2012 PST sample Sep j j Detect Results oN Sat 11 56AM 12 01 PM 9 53 PM oN por Saat 1 10 PM z 10 02 PM 2 r Fa oe E ri 5 ry oj j f ate r t 5 5 5 O w a oe a he Le hari ri ra merir nm ee dal z a 3 37 PM 13 PM 11 22 PM Ti aT T wT n EP 1 i 2 J 3 I E z ae 3 37 PM A 4 51PM 10 04PM 10 30PM 12 39 AM ay rA re s 6 05PM 10 58 PM 11 24PM 1 33AM Ti aT T T 1 1 Cad 2 J 3 ae 6 05 PM 09 Pl 7 18 PM any 12 05 AM i 0 70 30 A 7 19 PM oa 8 32 PM nae ee ape 03 a i O 7 30 A 7 37 Ph 9 46 PM 1 09 AM 1 36 AM Ad AN r Irr a z m m z The run file name path directory location and assay used is displayed along with instrument serial number and the run start complete date and time To go to the run file directory location Double click the path hyperlink or right click and select Open Directory Compass Software for Peggy User Guide page 112 Chapter 6 Run Status To copy the path Right click on the path hyperlink and click Copy The path can then be copied into document
199. iles Select File from the main menu and click Close All Compass Software for Peggy User Guide page 348 Chapter 9 Charge Assay Data Analysis Compass Analysis Settings Overview Compass has a variety of analysis features and settings that users can modify as needed to enhance run data To access these settings select Edit in the main menu and click Analysis If more than one run file is open select the run file you want to view analysis settings for from the list Cut Copy Ctrl C Paste Ctrl V Analysis Training data_Hi Low ERK cbz Preferences DemoData_Chargqe_HeLa_ERK12 cbz Advanced Images Analysis Settings Analysis Settings Advanced Peak Fit Advanced cee eae Peak Names Standards Standards Peak Width Allowable Drift Sample Peak Fit Starting Width Ratio ae Advanced Median Filter Threshoki Ratio Override Median Filter Threshold Limit Image Remove Restore Original Compass Software for Peggy User Guide Compass Analysis Settings Overview page 349 To move between analysis pages in this window click on an option in the list on the left The following anal ysis settings can be user customized in Compass Advanced Lets you customize analysis settings for samples standards and image data Images Lets you view the chemiluminescent exposures taken during the run and view data for dif ferent exposures in the Analysis screen Peak Fit Lets you customize peak fit sett
200. illaries blocking reagent names or primary antibody names and attributes in the run data NOTE Analysis settings are run file specific However settings can be imported or exported for use with other run files For more information see Importing and Exporting Analysis Settings on page 261 Peak name groups are displayed in the analysis settings box Analysis Settings Peak Names 1 The Peak Names group shown is a Compass template group Users can make changes to this group and cre ate new groups To view settings for a group click on the group name in the analysis settings box Creating a Peak Names Group 1 Select Edit in the main menu and click Analysis then click Peak Names in the options list 2 Click on the Peak Names 1 template group in the analysis settings box Analysis Settings Peak Names 1 3 Enter anew name for the group Compass Software for Peggy User Guide page 240 Chapter 8 Size Assay Data Analysis Analysis Settings 4 Click in the first cell in the Name column in the analysis settings peak table Analysts Settings EREL 2 Name MW Color Range Show EA y 100 N 0 5 Enter a sample protein name associated with the primary antibody used in the run Analysis Settings EREL 2 Name MW Color Range Show ERK 100 ae 6 Click in the first cell in the MW column Analysis Settings EREL 2 Name MW Color Range Show ERK2 00 D io 7 Enter the molecular wei
201. in the graph area kx Graph gt Image Lane EB E Samples 1300 ERK2 C1 10 Chemilumines cence 12 Ap 6 0 116 180 MW kDa Plot Labels You can customize the plot labels displayed on the electropherogram with these options Plot Label Sample Attribute Primary C Attribute a Go Capillary Exposure Plot labels are shown on the right side of the graph pane Sample Checking this box will display the sample name If sample names were entered in the assay template Assay screen those names will display here Otherwise Sample default name will display Primary Checking this box will display the primary antibody name If primary antibody names were entered in the assay template Assay screen those names will display here Otherwise Primary default name will display Capillary Checking this box will display the cycle and capillary number For example C1 3 indicates cycle 1 capillary 3 Attributes Checking this box will display attribute text If attribute information was entered for sam ples or primary antibodies in the assay template Assay screen they can be selected as plot labels Compass Software for Peggy User Guide Changing the Electropherogram View page 209 Exposure Checking this box will display the exposure time s used for the data The following example shows an electropherogram with all plot labels selected Chemilumine
202. ing Non admin Users Add a user to the server to allow that user to log in to Compass To do this 1 Select Users from the Site Administration home page Site administration Prote x W 1 gt 5 10 1 2 18 8000 admin Welcome admin Change password Log out Site administration i Recent Actions i Add Change My Actions Add Change steveg User WF steveg User TypeScientist User TypeScientist User a TypeScientist User TypeReviewer User P TypeReviewer User fay 2 From the Users page select Add User Compass Software for Peggy User Guide Authorization Server gt amp 10 1 2 18 8000 admin auth user Home Auth gt Users Select user to change qi e Username Email address First name Last name fay fay deng k k k com qc Gallagher Nguyen 3 Fillin the fields to create a new user gt C B 10 1 2 18 8000 admin auth user add Home Auth gt Users gt Add user Add user Staff status First enter a username and password Then you ll be able to edit more user options Username i Required 30 characters or fewer Letters digits and y _ only Password Password confirmation Enter the same password as above for verification By staff status All Yes No By superuser status All Wes No By active All Yes No By groups All Administrator page 413 Save and add another Sa
203. ing rows in the experiment pane to make sure all standards are identified correctly Step 3 Checking Capillary Registrations All capillaries should have a single capillary registration peak To verify the registrations are identified cor rectly Compass Software for Peggy User Guide page 300 Chapter 9 Charge Assay Data Analysis 1 Click the Analysis screen tab 2 Click Show Registrations and Single View in the View bar 3 Click the Graph tab 4 Click on the first row in the experiment pane The registration peak will be the first and largest peak in the electropherogram Check that the two registration peaks are identified and labeled Reg 1 and Reg 2 in the electropherogram They will also be identified with a purple R in the peaks table DemoData_Charge_HeLa_ERK12 Compass o a File Edit View Instrument Window Help tJ fg E Assay TE Run Summary m Experiment ml I lt Gre 7 T gg W A Sample Primary Cycle Registrations High phos ERK1 2 Low phosp ERK1 2 High phos ERK1 2 Low phosp ERK1 2 High phos ERK1 2 Low phosp ERK1 2 High phos ERK1 2 Low phosp ERK1 2 High phos ERK1 2 Low phosp ERK1 2 High phos ERK1 2 Low phosp ERK1 2 High phospho HeLa in 5 8 C1 1 Reg 73 k b ee ee d il Fluorescence 450 500 550 600 650 700 750 800 850 900 950 1 000 Position FRA Peaks EE Capillaries Sample Primary Cap Peak Position High
204. ings Click OK to save changes and exit Click Cancel to exit without saving changes Standards Analysis Settings Groups Standards settings are saved as a group and multiple settings groups can be created Specific group settings can then be applied to individual capillaries sample names or attributes in the run data NOTES ProteinSimple recommends using the Compass default values for standards analysis settings These set tings are included in the default Standards group Analysis settings are run file specific However settings can be imported or exported for use with other run files For more information see Importing and Exporting Analysis Settings on page 261 Standards groups are displayed in the analysis settings box Analysis Settings Standards Add Remove The Standards group shown contains the Compass default settings Users can make changes to this group and create new groups To view settings for a group click on the group name in the analysis settings box Changing the Capillary Used for the Ladder Known ladder standards are used to calculate the molecular weights of unknown sample proteins Capillary 1 is typically used for ladder However users can change the ladder capillary as needed or opt to not use a ladder at all Compass Software for Peggy User Guide Standards Settings page 251 NOTES When the ladder capillary is set to none fluorescent standards information is used to cal
205. ings for sample data Peak Names Lets you enter custom naming settings for sample proteins associated with specific primary antibodies or attributes and have Compass automatically label the peaks in the run data Standards Lets you customize the pl and positions Compass uses to identify fluorescent standards and registration peaks Compass Software for Peggy User Guide page 350 Advanced Analysis Settings Chapter 9 Charge Assay Data Analysis The advanced analysis settings page lets you view and change analysis settings for samples standards and image data To access these settings select Edit in the main menu and click Analysis then click Advanced in the options list NOTE Settings can be modified in an assay prior to starting a run or ina run file once the run has finished executing Analysis changes made to an executing run will not be saved to the final run file Advanced Images Analysis Settings Peak Fit Peak Names Standards remon Override Compass Software for Peggy User Guide Analysis Settings Advanced Standards Peak Width Allowable Drift Sample Peak Fit Starting Width Ratio Image Median Filter Threshold Ratio Median Filter Threshold Limit Restore Original Advanced Analysis Settings page 351 Click Import to import an analysis settings file This will be explained in more detail in Importing Analysis Settings on page 386 Click Export to export the current analysis s
206. ion in the Protocol or Template panes Exit Closes Compass Compass Software for Peggy User Guide page 22 Chapter 2 Size Assays Edit Menu The following Edit menu options are active Instrument Window Help Cut Copy Ctrl C Paste Ctrl V Default Analysis Analysis Preferences Copy Copies the information in the Protocol or Template panes into other documents Default Analysis Displays the default settings that will be used to analyze the run data generated with an assay Analysis Not active in this screen Preferences Set and save custom preferences for data export plot colors in the graph and Peggy s Twitter settings See Chapter 10 Setting Custom User Preferences for more information Compass Software for Peggy User Guide Reagent Color Coding page 23 Reagent Color Coding The Assay screen uses color coding to identify various assay reagents in all panes The Layout pane is shown in the following example __Separation Matrix Stacking Matrix TOFErZL ToOAmMeoaaE Pink Samples and Ladder Dark Blue Blocking reagent Antibody Diluent Orange Primary antibody Gold Secondary HRP conjugate Yellow Luminol Peroxide mix Nocolor coding Separation Matrix clearly designated Nocolor coding Stacking Matrix clearly designated e Light Blue Water dispensed around Separation and Stacking Matrices Compass Software for Peggy User Guide page 24
207. isplay here Otherwise Primary default name will display Secondary Ab Secondary antibody name If a secondary antibody name was entered in the assay template Assay screen those names will display here Otherwise Secondary default name will display Capillary Cycle and capillary number For example C1 3 indicates cycle 1 capillary 3 NOTE Peggy runs up to eight cycles 12 capillaries at a time so the cycle number displayed will always be C1 C8 depending on the number of cycles programmed Attributes Attribute text If attribute information was entered for any of the rows or wells in the assay template Assay screen up to four can be selected and displayed as lane labels Compass Software for Peggy User Guide Changing the Virtual Blot View page 193 Viewing the Uncorrected Sample Baseline 1 Click the Remove Baseline button active for sample data only This will remove the automatic base line correction E Graph tE Image Lane D hi EF DE um 2 Click the Remove Baseline button again to return to the default baseline corrected view Overlaying Standards Data on Sample Lanes Data for the standards can be overlaid on the sample data in the virtual blot so users can view the raw unaligned uncorrected lane data To do this 1 Click the Overlay Standards Data button active for sample data only An overlay of the raw sample and standards data will display Compass Software for Peggy User Guide page
208. ithm to generate a 160 bit hash code that is unique for all files All files saved by Compass are encrypted with a digital key This key along with the hash codes guarantees the file history is correct and no other edits were made All changes saved to a file have the electronic signature of the user who saved the file The e Signature command allows a user to sign off on a state such as approved or verified There is no individual ownership of files all users who log into Compass can open any file Compass Software for Peggy User Guide page 420 Chapter 11 Compass Access Control and 21 CFR Part 11 Compliance Compass Software for Peggy User Guide
209. k pi Hide Name Add Peak Sample Primary Cap Peak Name Position Height Area Area Widt Add Baseline Point Low p ERKL 2 33 0 034 Clear All To add an unidentified peak to the data Right click the peak in the electrooherogram or peaks table and select Add Peak Compass will calculate and display the results for the peak in the results tables and identify the peak in the electropherogram NOTES To remove sample peak assignments that were made manually and go back to the original view of the data right click in the electropherogram and click Clear All Virtual blot data in the lane pane will also update to reflect changes made in the graph pane Compass Software for Peggy User Guide page 314 Chapter 9 Charge Assay Data Analysis Hiding Sample Data You can hide the results for a sample protein in the results tables without completely removing it from the reported results To to this 1 Click Show Samples in the View bar 2 Click Single View in the View bar 3 Click on the row in the experiment pane that contains the sample you wish to correct then click the Graph tab 4 Right click the peak in the electrooherogram or peaks table and select Hide peak Compass will hide the peak data in the results tables ha Graph tE Image eae EF i a _ 1400 ppErki1 pErk Erk1 pERK2 mi 1300 ppERK2 1200 1100 _ 1000 S S 500 a 800 E 700 E 600 4500 400
210. k Analysis in the options list Analysis Graph Twitter Export Standards Export using a comma as the column deliminator Export Standards Selecting this option includes data for the standards in each sample when run data is exported When this option is deselected only sample data will be exported This option is selected by default Export using a comma as the column delimiter Selecting this option exports run data in csv for mat When this option is deselected the data is exported in txt format Click Apply to apply changes to any open run files in Compass Click Restore Defaults to restore Compass default settings Click OK to save changes and exit e Click Cancel to exit without saving changes Compass Software for Peggy User Guide page 392 Chapter 10 Setting Custom User Preferences Selecting Custom Plot Colors for Graph Overlay Select Edit in the main menu and click Preferences then click Graph in the options list type filter text Graph Analysis Graph Twitter Plot color 1 Apply colors to stacked plots Plot color 2 Plot color 3 Plot color 4 Plot color 5 Plot color 6 Plot color 7 Plot color _ Cc _ _ _ m Plot color 9 Apply colors to stacked plots Selecting this option applies the color scheme to individual electro pherograms when the Stack the Plots option is selected in the Analysis screen graph pane NOTE If Apply colors t
211. l state provincial or national environmen tal and health regulations Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste vial before you store han dle or dispose of chemical waste Compass Software for Peggy User Guide page 60 Chapter 3 Running a Size Assay on Peggy Stopping a Run 1 To stop a run click Stop When the run stops instrument status will go to Not Ready and a Cleanup but ton displays a NOTE Ifa run is stopped prior to completion Peggy must perform a cleaning protocol to remove used Not Ready Cleanu capillaries from system trays This is required to prepare the system for the next run 2 Click Cleanup Cleaning Cleaning Stop Wed 3 48 PM Wed 3 56 PM P Allow Peggy to complete the cleaning protocol which takes about ten minutes When complete instru ment status will change to Ready and a new run can be started Compass Software for Peggy User Guide page 61 Chapter 4 Charge Assays Chapter Overview Assay Screen Overview Reagent Color Coding Opening an Assay Creating a New Assay Making Changes to an Existing Assay Switching Between Open Assays Creating a Template Assay Viewing and Changing the Detection Exposures Copying Protocols and Templates Printing Protocols and Templates Importing and Exporting Protocols and Templates Compass Software for Peggy User Guide page 62 Chapter 4 Charge Assay
212. l also be identified with a green S in the peaks table To view band labels roll the mouse over the individual bands Compass Software for Peggy User Guide Checking Your Results page 167 File Edit View Instrument Window Help ees 2m g Eeey Ctunsummay Primary Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 Zi 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 A ao 6 rm Graph 2 Image E Lane A G n g p E Melso F lt ie x i E E a lt lt lt P lt Ci lt lt lt s amp oi a lt V amp lt amp amp amp amp amp Sa re HH Peaks Sample biotin S biotin biotin biotin S biotin biotin biotin biotin S biotin hela Shela EE Capillaries Primary Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Rlacki Cap Ci ci 1 Ci Ci Git Peak VvrFwWoOOnN DU amp WN
213. l be able to Read Tweets from your timeline See who you follow and follow new people www proteinsimple com Compass from ProteinSimple ti POR YAU prome Cancel and return to app Post Tweets for you Username or email Password Remember me Forgot password Authorize app No thanks This application will not be able to Access your direct messages See your Twitter password You can revoke access to any application at any time from the Applications tab of your Settings page By authorizing an application you continue to operate under Twitter s Terms of Service In particular some usage information will be shared back with Twitter For more see our Privacy Policy 2 Enter a user name or email and password then click Authorize app A new page will display in the browser with a PIN number 3 Enter the PIN number in the set account window in Compass and click OK Compass Software for Peggy User Guide page 396 Chapter 10 Setting Custom User Preferences Enter PIN given by Twitter Web Site 4 The user name will now appear in the Twitter User Name box Select one or all of the Peggy tweet options in the Tweet When box then click Apply i Preferences Analysts Graph Twitter User Name Twitter Tweet When C Run is started E Run is completed C Errors Tweet Message 5 To confirm the Twitter account is receiving messages click Tweet Message
214. laying run data in the graph image and lane panes in a per capillary single format or up to 96 capillary format This view is selected in the View bar or the View menu geerzEl E Instrument Window Single View Multiple View Standards Registration Samples Filter View Region Show Hidden Capillary view buttons in the View bar E Single View Multiple View Compass Software for Peggy User Guide page 156 Chapter 8 Size Assay Data Analysis To view data ina per capillary format Click Single View in the View bar or select View in the main menu and click Single View Fil oo Help i b assay CJ Run Summary os AW HeLa HeLa HeLa Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa Biotin Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Mie t w w w wwwnnnnnn Nn NN NN NR tu gt e e FFF eww Ww Ww WwW Graph mage E tane H Ha HeLa C1 6 HeLa C1 9 HeLa C1 7 ce Chemiluminescen oPSSSS8 58888 88888 588 Cap Peak Name P
215. le Sample Primary Cap Peak Name Position MW Height Area o Area Width Biotinylat Blocking C11 5 Ldr1i6 3175 2 59215 Biotinylat Blocking Ldr 180 3715 5 78654 Peaks that Compass names automatically using the user defined peak name analysis parameters are NOTES color coded The reported molecular weight for immunodetected sample proteins in Compass may vary slightly from predicted molecular weights based on sample and assay conditions To view all rows Click the Peaks tab then use the scroll bar or click Maximize in the upper right corner Toresize columns Click the Peaks tab Roll the mouse over a column border until the sizing arrow appears then click and drag to resize Peak table column descriptions are as follows Sample Sample name If sample names were entered in the assay template Assay screen those names will display here Otherwise Sample default name will display e Primary Primary antibody name If primary antibody names were entered in the assay template Assay screen those names will display here Otherwise Primary default name will display e Cap Cycle and capillary number For example C1 3 indicates cycle 1 capillary 3 NOTE Peggy runs up to eight cycles in an experiment 12 capillaries at a time Capillary and cycle numbers are displayed in the Experiment tab Peak Peak number Peaks are numbered in order of detection Compass Software for Peggy User Guide page
216. le Primary Cycle Cap S 1 2 3 HH Peaks FE Capillaries Sample Primary Cap Peak Name Position MW kDa Height Area Area Width S N Compass Software for Peggy User Guide Software Overview page 3 Changing the Screen View To move between the Assay Run Summary and Analysis screens just click the button in the screen tab located in the upper right corner of the main window E Assay Run Summary gE Analysis Assay Screen The Assay screen is used to create view and edit assays Users can assign well locations for assay plate reagents modify assay protocol steps enter assay notes and add annotations for individual wells on the assay plate Multi Western Compass File Edit Instrument Window Help Pg Run Summary gfe Analysis Protocol N E Notes a Add v Remove eee ne eee eee eee OOOO OO OO OO OO a SS m Layout AO SOI as Cyclel Cycle 2 Cycle3 Cycle4 Cycle5 Cycle6 Cycle7 Cycle8 Separation Matrix Stacking Matrix Sample Separation Time min 40 0 40 0 40 0 40 0 40 0 40 0 40 0 40 0 TA Separation Voltage volts 250 250 250 250 250 250 250 250 b Matrix Removal p Blocking Time min 25 0 25 0 25 0 25 0 25 0 25 0 25 0 25 0 a Primary Ab Time min 120 0 120 0 120 0 120 0 120 0 120 0 120 0 120 0 My Secondary Ab Time min 60 0 60 0 60 0 60 0 60 0 60 0 60 0 60 0 M Detection N o P Template m Edit
217. lect a name to apply group settings to all capillaries that use this sample name in the run file Attributes All sample attributes entered in the assay template Assay screen will display in the drop down list Select an attribute to apply group settings to all capillaries that use this attribute in the run file Cycle 1 8 When this option is selected group settings will be applied to all capillaries in the cycle Custom settings Lets you choose specific capillaries to apply the group settings to When this option is selected the following pop up box appears to let you enter a specific capillary number or range of capillaries Enter cycle and capillary descriptor Examples 3 1 1 12 Cycle 2 capillary 3 and cycle 1 capillaries 1 through 12 1 3 6 8 10 Cycles 1 through 3 capillaries 6 8 and 10 Cancel 6 Ifyou need to change the analysis group used for a data set click the cell in the Settings column and click the down arrow Select a group from the drop down list Compass Software for Peggy User Guide Importing and Exporting Analysis Settings page 261 Override Apply To Settings Biotinylated Ladder Standard Standard K New Standards 7 Repeat the previous steps to apply other groups to specific run data 8 To remove a data set click on its cell in the Apply To column then click Remove 9 Click OK to save changes Importing and Exporting Analysis Settings The analysis settings i
218. lick Preferences then click Twitter in the options list ty type filter text Analysis Graph Twitter User Name Te Tweet When E Run is started E Run is completed E Errors Tweet Message Click Apply to apply changes e Click Restore Defaults to restore Compass default settings Click OK to save changes and exit Click Cancel to exit without saving changes To have Peggy tweet a Twitter account NOTES To set Peggy up to tweet the computer you are using must have an internet connection To tweet Peggy must be connected to the internet through a network connection or via the local lab computer Compass Software for Peggy User Guide Setting Peggy Up to Send Tweets page 395 ProteinSimple recommends setting up a separate Twitter account for Peggy This lets multiple people in the lab follow run progress It also lets users send tweets directly from Peggy to all users for example to notify others when the instrument is available or when an error has been reset etc 1 Click Set Account A set account window will display in Compass and the following browser window will open sje Js NE Y http api twitter D X amp Y hamer eBay W Twitter Authorize an appli int We OS x Convert x F Select x Google v J Search More SignIn sis gmail Email from Google Authorize Compass to use vy your account Compass By ProteinSimple This application wil
219. lick on the first row in the experiment pane then click the Graph tab Check that the electrophero gram has three standard peaks labeled Std 1 0 Std 12 0 and Std 180 0 They will also be identified with a green S in the peaks table File Edit i reran Window Help HEA Hs amp Fy Assay Py Run Summary EE Esperin Show Registrations 8 Graph Te O 72 Sample Primary Cycle Standards biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki a BEZBESSESESEE Fluorescence oS SB asg ERAEN 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1 000 Position Sample Primary Cap Peak Position Height S biotin Blocki i 393 3 biotin Blocki 2 35 biotin Blocki 4 2 S biotin Blocki 27 2 biotin Blocki 5 0 biotin Blocki
220. lick the cell in the value col umn next to Well Row and select a different row on the assay plate T Protocol Add Remove Cycle 1 Sample Separation Immobilization Time sec 100 0 Primary Ab Time min 120 0 Well Row B1 ki Load Time sec B1 Washes Wash Load Time sec 20 0 Wash Soak Time sec 150 0 Secondary Ab Time min 60 0 Tertiary Ab Time min 10 0 Detection NOTE Only rows designated as primary antibody in the Layout tab can be selected in the Well Row drop down menu 5 If needed change the secondary incubation time To do this click the cell in the value column next to Secondary Ab Time min and enter a new value in minutes Compass Software for Peggy User Guide Creating a New Assay page 75 Add Remove Cycle 1 b Sample p Separation bt Immobilization Time sec 100 0 t Primary Ab Time min 120 0 4 Secondary Ab Time min 60 0 Well Row Cl Load Time sec 2 0 Washes 2 Wash Load Time sec 20 0 Wash Soak Time sec 150 0 b Tertiary Ab Time min 10 0 b Detection 6 If needed change the secondary incubation reagent row location To do this click the cell in the value column next to Well Row and select a different row on the assay plate Protocol gt Te Not Add Remove Sample Separation Immobilization Time sec 100 0 Primary Ab Time min 120 0 Secondary Ab Time min 60 0 Well Row Cl Load Time sec Cl Washes Wash
221. ll display If this occurs click Yes to cancel the run and perform the manifold cleaning Compass Software for Peggy User Guide page 54 Chapter 3 Running a Size Assay on Peggy 3 Page 1 Check Water and Waste The fluid levels in the accessory module bottles will be checked by the software If the levels in both bottles will allow Peggy to complete the run the wizard screen will dis play Water Level OK and Waste Level OK messages Click Next to proceed Water level OK Waste level OK r Start Run EE Check water and waste Make sure there is enough water and room in the waste bottle The run will be terminated if the water bottle empties or the waste bottle fills during the run NOTE If the waste level is too high or the water level is too low to complete the run messages to indicate one or both will be presented in this screen If this occurs fill or empty each bottle as indicated using the procedures outlined in the Peggy User Guide When this is complete the error status will be automatically updated and allow the Start Run Wizard to proceed Compass Software for Peggy User Guide Starting a Run page 55 4 Page 2 Replace Sponge A new sponge should be used each time a new experimental run is started Discard the old sponge and put a new sponge in the manifold wash station a Start Run X Replace Sponge Put a new sponge in the manifold wash station
222. llowing example shows an electropherogram with all plot labels selected pE mage E kene IER ppErk1 pErk1 Erk pERK2 ERK ppERK2 High phospho HeLa in 5 8 1 mg mL ERK1 2 Goat antitabbit HRP Exposure 240 0s C1 1 u TI T TI a E 3 E T O 200 70 700 650 600 10 500 450 400 ao 300 2a 200 150 100 30 D4 s0 Si 52 53 54 55 56 57 58 59 G0 Gi 62 63 64 65 G6 67 BS 69 70 71 pl Compass Software for Peggy User Guide Changing the Electropherogram View Selecting Data Viewing Options page 339 The graph view menu provides multiple user options for changing what type of electropherogram data is displayed To access the view menu click the down arrow next to the graph pane toolbar in Graph a E Image Lane 1t ef E Samples ppErk1 pErk1 Erk pERK ERK2 ppERK am Hi phospho He gt a ERK14 E 2 700 z 600 E 0 a O 400 20 j 100 co 50 51 52 53 54 55 56 57 58 59 60 61 62 63 G4 65 66 67 68 639 pl K Bi 7 0 Sample Sample Baseline Corrected Baseline Fit Baseline Points Fit Fit Baseline Corrected Standards Baseline Corrected Smooth A check mark next to the menu option indicates it is currently selected Multiple options can be selected at a time NOTE Unless otherwise noted graph view menu options are available for sample data only Compass Software for Peggy User Guide page 340 Chapter 9 Charge Assay Data Analysis Sa
223. lly used for the registration New Standards Fluorescent Peaks Registration NOTE In order for Compass to perform data analysis at least one peak must be selected for registration 10 Select which standards should be used for molecular weight determination of sample proteins by click ing the checkbox in the Fit column The standards not used for registration are typically used for fit New Standards Fluorescent Peaks Position Fit Registration Compass Software for Peggy User Guide Standards Settings page 255 11 Click the arrow in the drop down list next to Ladder Capillary then click a capillary number or none from the list Capillary 1 is typically used for the ladder Ladder Capillary Aone 1 MW Positia 1 20 40 50 s 66 60 90 707 116 go l g 180 90 i 11 12 Add Remove Compass will use the data in the selected capillary to calculate the molecular weights for sample pro teins using the information in the ladder table If none is selected Compass will instead use the informa tion in the fluorescent peaks table fluorescent standards to calculate molecular weight for sample proteins NOTES When the ladder capillary is set to none the ladder table becomes inactive and cannot be modified The reported molecular weight for immunodetected sample proteins in Compass may vary slightly from predicted molecular weights based on sample and assay conditions 12 Ifa ladder capillary was selec
224. lot View page 191 Inverting the Virtual Blot 1 Click the Invert button The virtual blot image will invert E Graph 1 Image Lane D in Be b Invert 2 Click the Invert button again to return to the default view Selecting Lane Labels The labels shown above the lanes in the virtual blot can be customized To do this 1 Click the Edit Labels button The label box will display Compass Software for Peggy User Guide page 192 E Graph Image Lane Chapter 8 Size Assay Data Analysis Sig Do O g Lane Label a T e e E LHE W Sample E Attribute r tie e E ee gem __ the Sf AL soe eee i ow ow o F o oo oe ow Oo or a C Primary Ab T Attribute C Secondary Ab Attribute T Tertiary Ab Attribute W Capillary 180 116 E 30 E 65 2 Check one or multiple label boxes and uncheck those you don t want to display To remove labels com pletely uncheck all boxes The following label options are available Sample Sample name If sample names were entered in the assay template Assay screen those names will display here Otherwise Sample default name will display Blocking Blocking reagent name If a name was entered in the assay template Assay screen that name will display here Otherwise Blocking default name will display Primary Primary antibody name If primary antibody names were entered in the assay template Assay screen those names will d
225. ltiple copies of the server may be run on the same network and each server will have its own user database To enable Compass to use a particular Authorization Server click Edit then Preferences and Access Con trol and enter the server IP address using format X X X X Compass Software for Peggy User Guide Authorization Server page 411 NOTES Always use the default port setting of 8000 this should not be changed If the server is installed on the same computer as Compass e g the local machine enter LocalHost instead of the IP address Contact your local IT Administrator to assist with installing the Authorization Server in your preferred format Server Administration The Authorization Server is configured through a web interface at the IP address of the server on port 8000 To access the Server home page open any browser and type the IP address on port 8000 in a X X X X 8000 or http X X X X 8000 format Use LocalHost instead of the IP address if the Server is installed on the local machine The default server administrator is User admin Password admin After installing the Authorization Server the administrator user name and password can be changed Login ProteinSimplesite x 1 gt 5 10 1 2 18 38000 admin ProteinSimple Administration Username admin Password Log in Compass Software for Peggy User Guide page 412 Chapter 11 Compass Access Control and 21 CFR Part 11 Compliance Add
226. mary Cycle Sample Primary Sample Primary Sample Primary Sample Primary Sample Primary Sample Primary ries sure 1 Sampie xX Hide Sample H Clear All Data for selected rows will be hidden in all data views and results tables except for the image pane To view hidden rows Select View in the main menu and click Show Hidden Hidden rows will become visible again in all panes and hidden rows will be marked with an X in the experiment pane E Experiment O a Sample Primary Cycle X Sample Primary Sample Primary Sample Primary Sample Primary Sample Primary X Sample Primary ee 1i Sample Primary e To unhide rows Select the hidden row s Right click on one of the selected rows and click Unhide Setting Run Data Display Filters The filter lets you auto hide specific capillaries in the run file data or only show data for peaks that Compass identified automatically using the user defined peak name analysis parameters NOTE When more than one run file is open filter settings will be applied to all files e To filter data to show specific capillaries only Select View in the main menu and click Filter Uncheck the boxes for the capillaries you do not want shown then click OK Compass Software for Peggy User Guide Viewing Run Data page 161 Fiter Capillaries SSE SSE SS RRA 8
227. more than one peak label option is selected peak name labels will always be used for named peaks The reported molecular weight for immunodetected sample proteins in Compass may vary slightly from predicted molecular weights based on sample and assay conditions Compass Software for Peggy User Guide Changing the Electropherogram View page 205 b cee Chemilumines cence mal 200 100 D0 900 200 700 600 500 400 300 avail o amp z 116 Baseline and Grid Options You can view the calculated baseline fit peak integration and show grid lines with these options Ah Fitted Peaks J Baseline Fit Fitted peaks Checking this box will display how the peaks were fit by the software NOTE This option is only available for sample data Compass Software for Peggy User Guide page 206 Chapter 8 Size Assay Data Analysis TE u D p wa a E 3 E D D 000 200 200 700 600 500 400 300 200 So 8 Baseline Fit Checking this box will display the calculated baseline for the peaks Baseline points will also display for regions of the electrooherogram considered to be at baseline Compass Software for Peggy User Guide Changing the Electropherogram View page 207 NOTE This option is only available for sample data Compass Software for Peggy User Guide page 208 Chapter 8 Size Assay Data Analysis Grid Lines Checking this box will display grid lines
228. mple Clicking this option will display raw uncorrected sample data ppErk1 pErk1 Erk1 ppERK Hi shospho HeLa C1 1 5 E segs 8 8 Chemilumines cence 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 pl Sample Baseline Corrected Clicking this option will display sample data with the baseline sub tracted zeroed This is the default view ppErk1 pErk1 Erk1 ppERK Hi phospho HeLa 1 1 ow o D o ws a c E 3 E D a 1000 S00 200 700 600 500 7 400 300 200 50 51 52 53 54 55 56 57 58 459 60 61 62 63 64 65 66 67 68 69 70 pl Compass Software for Peggy User Guide Changing the Electropherogram View page 341 Baseline Fit Clicking this option will display the calculated baseline for the raw sample data In the example that follows both Baseline fit and Sample are selected NOTE This option is selected automatically when Baseline Fit is selected in graph options pErk1 ppERK2 Erk1 Hi phospho HeLa C1 1 a TI T TI wn a E 3 E D D 5650 5 675 5700 5 725 5 750 5 775 5 900 5 825 5850 5 875 5 900 5 925 5 950 5975 6 000 6 025 6 050 6 075 6 100 pl Baseline Points Clicking this option will display regions of the electropherogram considered to be at baseline In the example that follows both Baseline Points and Sample are selected Compass Software for Peggy User Guide page 342 Chapter 9 Charge Assay Data Analysis NOTE Thi
229. mple biotin hela hela hela hela hela hela hela hela hela hela biotin hela hela hela hela hela hela hela hela hela hela hela biotin hela hela hela hela hela hela hela hela hela hela hela biotin hela hela hela hela hela hela Primary Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki A Cycle gt 22 A F amp F FP FP FH HWW swwnwwnwnswwnwnwnwnwnnin nn nN NNN NN NK FF RP RP RP RP RR RP eR eR ea Graph i Image Lane JT HO n Registrations H Fluorescence N A U O N O 450 500 550 600 650 700 750 800 850 900 950 1 000 Position HE Peaks N EH Capillaries Primary Blocki Blocki Blocki Blocki Cap C2 10 C2 10 C2 10 2 10 Peak Position 1 2 3 4 259 283 302 331 Height 21 15 5 12 18 Compass Software for Peggy User Guide Checking Your Results page 169 If the registration peak is not identified correctly right click the correct registration peak in the electro oherogram or peaks table and select Re
230. mples Exp 30 0 W Compass Software for Peggy User Guide page 142 Chapter 8 Size Assay Data Analysis Lane Pane Virtual Blot Like Image Data Click the Lane tab to view data for immunodetected sample proteins fluorescent standards or capillary reg istrations as bands in individual lanes This virtual blot like image is similar to traditional blot results Data for samples in the lane view is shown in the following example and immunodetected proteins are displayed as bands EE Graph 3 Image Lane Oo KE g hoo MW kDa 43 Area 21641 To view information for a band roll the mouse over a band until the info box appears NOTE The reported molecular weight for immunodetected sample proteins in Compass may vary slightly from predicted molecular weights based on sample and assay conditions Lane data displayed in the virtual blot is automatically aligned by Compass To view raw unaligned lane data and learn more about virtual blot viewing options see Changing the Virtual Blot View on page 190 Compass Software for Peggy User Guide How Run Data is Displayed in the Analysis Screen page 143 Peaks Pane Calculated Results Click the Peaks tab to view tabulated results for immunodetected sample proteins fluorescent standards or capillary registrations Each row in the table shows the individual results for each peak detected in each cap illary Data for samples in the peaks table is shown in the following examp
231. n Place the lidded 384 well plate on the cooling block in the tray ensuring that the A1 well position is aligned with the upper left corner of the cold block When this is complete click Next The sample tray will close Start Run Load Sample Plate Remove the old sample plate and load the new plate 1234567 8 91011 12131415617 81921224 wozezrxc xranmoomD A1 Position Compass Software for Peggy User Guide page 58 Chapter 3 Running a Size Assay on Peggy NOTES Peggy requires that plate lids be used on sample plates Ifa lid is not detected a message will be displayed in the Start Run Wizard Compass software will reopen the sample tray to allow the user to insert a lid When inserting the sample plate ensure that the plate is firmly seated and level on the cold block Plates that are not level can interfere with the movement of the sample tray 7 Page 5 Data File The data file name will automatically default to the assay name appended with the current date and time To change the file name begin typing in the text box To change the directory where the data file will be stored click Browse Start Run Xs Begin the Automated Run The following protocol will be run You can change the results location and prefix Assay Multi Western_Hela HUT78 Cycles 8 Schedule Overlapping with hold Run name 2012 05 31_09 29 06_Multi Western_Hela HUT78 Browse Location C U
232. n a run file can be exported as a separate file This allows the same analysis settings to be imported into other assays or run files at a later time rather than having to re enter settings manually Importing Analysis Settings NOTE Importing an analysis settings file populates the settings in all Analysis pages 1 Open the run file or assay you want to import analysis settings to Select Edit in the main menu and click Default Analysis Assay screen or Analysis Analysis screen Click Import on any page a eh Select a settings file settings and click OK The imported settings will display in all analysis pages Exporting Analysis Settings NOTE Exporting an analysis settings file exports the settings in all Analysis pages Compass Software for Peggy User Guide page 262 Chapter 8 Size Assay Data Analysis 1 Open the run file or assay you want to export analysis settings from 2 Select Edit in the main menu and click Default Analysis Assay screen or Analysis Analysis screen 3 Click Export on any page The following window displays x ITE documents My Documents Compass Assays wtp Sexchanoye O a Organize v New folder Fr Favorites Documents library Arrange by Folder ME Desktop Assays ib Downloads Recent Places Name Date modi Type _ ERK analysis settings settings 10 16 201 SETTINGS WE Desktop _ default settings 10 16 201 SETTINGS Libraries E3 Documents E My Do
233. n uses color coding to identify various assay reagents in all panes The Layout pane is shown in the following example VOZErF AC lt TH nmo 0T P mS TOFZEr SLC ToHAmooop Pink Samples Blue Primary antibody Orange Tertiary antibody Gold Secondary HRP conjugate Yellow Luminol Peroxide mix Compass Software for Peggy User Guide page 66 Chapter 4 Charge Assays Opening an Assay To open an existing assay 1 Select File in the main menu and click Open Assay Edit Instrument Window Help New Assay Open Assay MEK assay Protein Test Save Save As Peggy Charge Peggy Charge 1017 Import Proteceol Erk Assay Import Template Export Protocol Export Template Print ee ee ODP Exit 2 Alist of the last five assays opened will display Select one of these assays or click Browse to open the Assay folder and select a different assay Compass Software for Peggy User Guide Creating a New Assay page 67 Creating a New Assay To create a new assay ProteinSimple recommends using the Peggy template assay and modifying from there as needed Step 1 Open a Template Assay 1 Select File in the main menu and click New Assay T Edit Instrument Window Help Mew Assay b Peggy Charge Open Assay Peggy Size Save Save As Import Protecol Import Template Export Protocol Export Template Print e 2 A list of template assays that can be u
234. ncubation rows can be deleted Rows for required assay reagents cannot be deleted Fim XiOB 123 465170 A Ja B B Cl c D D E 4 E F F NOTES Samples antibodies and blocking buffer can be dispensed in Rows A J and in columns 1 12 or 13 24 ProteinSimple recommends keeping two rows empty between the substrate and the closest dispensed HRP conjugate to avoid any interaction with the substrate Rows Kand L are purposely left empty do not pipet reagents into these rows of wells Compass Software for Peggy User Guide page 30 Chapter 2 Size Assays Separation and Stacking matrices must be dispensed in the designated wells Water is dispensed to reduce the potential of evaporation of the matrices Step 3 Modifying the Assay Protocol Optional 1 Click on the Protocol tab This pane displays the individual steps of the assay protocol and allows you to change parameters as needed When creating a new assay a default protocol will display which auto matically assigns all reagent locations for Cycles 1 8 Protocol T Notes Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle 8 b Separation Matrix gt Stacking Matrix gt Sample Separation Time min 40 0 40 0 40 0 40 0 40 0 40 0 40 0 40 0 gt Separation Voltage valts 250 250 250 250 250 250 250 250 gt Matrix Removal gt Blocking Time min 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 gt Primary Ab Time min 120 0 120 0 120 0 120 0 120 0
235. ne 9 melson Primary in Blocki Cycle Cap eo 1 3 3 Blockin 1 2 FPP PLP LP LP LS a oF VPP PP LP VP VP we FE PP 8 m oe Sr SF PS PEP OP HX PO PK PK KKH PSKP KF HF KF HM eK HMe OCKI Blocki 1 5 Blocki 1 6 Blocki 1 7 Block 1 8 e a a Sa E k aa aa aS aa ae Blocki 1 9 Blocki 1 Blocki 1 Blocki 1 ee se es Blocki 2 Blocki 2 Blocki 2 Blocki 2 Blocki 2 Blocki 2 SSS SS SSS ee See ee ee Se eo meaner Te 8 Se SS SS SS SS Blocki 2 Blocki 2 Blocki 3 Blocki 3 Blocki 3 Blocki 3 Blocki 3 Blocki 3 m t Rockin _ 2 EE Peaks EHF Capillaries 6 Blocki 3 c Blocki 3 Sample Primary Cap Peak Position Height a Blocki 3 SBiotin Block Cid 1 313 3214 E Blocki 3 Biotin Blocki ai 2 367 39 Blocki 3 SBiotin Blocki Cll 3 407 318 me Blocki 4 Biotin Blocki di 4 430 41 Blocki 4 Biotin Blocki Ci 5 563 31 Blocki 4 Biotin Blocki ai 6 797 60 Blocki 4 Biotin Blocki ai 7 826 43 Blocki 4 S Biotin Blocki dai g 866 27 Blocki 4 Biotin Blocki ci 9 902 27 Blocki 4 SHela _Blocki aa a 304 334 8 Hela Blocki a2 2 317 37 gt e E Compass Software for Peggy User Guide Viewing Run Data page 155 Switching Between Single and Multiple Views of the Capillaries You can switch between disp
236. nerates statistics across multiple runs No option selected When only one run is open groups will only contain capillaries from the same cycle When more than one run is open groups are created for each run Viewing Statistics Peak and Capillary Groups The Peak Groups pane reports statistics for each named protein in every group Each group shows the statis tics for named proteins which includes average area standard deviation CV and SEM The number in parenthesis after the sample name indicates the number of capillaries in the group Compass Software for Peggy User Guide page 306 Chapter 9 Charge Assay Data Analysis ee Capillary Groups Al Group Flot Sample Primary Hi phospho HeLa 24 ERK1 2 Primary 1 Hi phospho HeLa 24 ERK1 2 Primary 1 Hi phospho HeLa 24 ERK1 2 Primary 1 Hi phospho HeLa 24 EREL 2 Primary 1 Hi phospho HeLa 24 ERK1 2 Primary 1 Hi phospho HeLa 24 ERK1 2 Primary 1 Hi phospho HeLa 24 ERK1 2 Primary 2 Hi phospho HeLa 24 ERK1 2 Primary 2 Hi phospho HeLa 24 ERE1 2 Primary 2 Hi phospho HeLa 24 ERE1 2 Primary 2 Hi phospho HeLa 24 ERE1 2 Primary 2 Hi phospho HeLa 24 ERK1L 2 Primary 2 Low phospho HeLa 24 EREL 2 Primary 1 Low phospho HeLa 24 EREL 2 Primary 1 Low phospho HeLa 24 EREL 2 Primary 1 Low phospho HeLa 24 EREL 2 Primary 1 Low phospho HeLa 24 EREL 2 Primary 1 Low phospho HeLa 24 EREL 2 Primary 1 Low phosphe HeLa 24 EREL 2 Primary 2 Low phosphe HeLa 24
237. ng an Analysis Group 1 Select Edit in the main menu and click Analysis then click Standards in the options list 2 Click on the group in the analysis settings box you want to delete and click Remove Compass Software for Peggy User Guide Standards Settings page 259 Analysis Settings Standards New Standards 3 Click OK to save changes Applying Analysis Groups to Specific Run Data 1 Select Edit in the main menu and click Analysis then click Standards in the options list 2 Click on the group in the analysis settings box you want to apply to specific run data Analysis Settings Standards New Standards 3 Application of analysis groups to specific run data is done in the override box Click Add under the over ride box A default override data set will be created from sample information found in the run file Override Apply To Settings Biotinylated Ladder Standard Add Remove 4 Click the cell in the Apply To column then click the down arrow Compass Software for Peggy User Guide page 260 Chapter 8 Size Assay Data Analysis Override Apply To Settings tinylated Ladder Standard Biotinylated Ladde 5 Select an option from the drop down list This will apply the settings group selected to specific run data as follows Sample names All sample names entered in the assay template Assay screen will display in the drop down list otherwise the default name of Sample will be shown Se
238. ngs settings X Hide Folders Cancel 4 The default directory is Compass Assays Change the directory if needed 5 Enter a file name and click Save The settings will be saved as a settings file Compass Software for Peggy User Guide page 388 Chapter 9 Charge Assay Data Analysis Compass Software for Peggy User Guide page 389 Chapter 10 Setting Custom User Preferences Chapter Overview Custom Preference Options Setting Data Export Options Selecting Custom Plot Colors for Graph Overlay Setting Peggy Up to Send Tweets Compass Software for Peggy User Guide page 390 Chapter 10 Setting Custom User Preferences Custom Preference Options Users can set and save custom preferences for data export plot colors in the graph and Twitter communica tion To access these settings select Edit in the main menu and click Preferences Analysis Export Standards Export using a comma as the column deliminator C Restore Defaults To move between preferences pages in this window click on any option in the list on the left The following items can be user customized in Compass Analysis Lets you customize data export options Graph Lets you customize graph color displays e Twitter Lets you configure Peggy to Tweet run status Compass Software for Peggy User Guide Setting Data Export Options page 391 Setting Data Export Options Select Edit in the main menu and click Preferences then clic
239. ngs box Analysis Settings Peak Names 1 Add Remove 3 Enter anew name for the group Compass Software for Peggy User Guide Peak Names Settings page 369 Analysis Settings ERKI 4 Clickin the first cell in the Name column in the analysis settings peak table Analysis Settings ERKI Color Range Show E 00 5 Enter a sample protein name associated with the primary antibody used in the run Analysts Settings ERKI Name pl Color Range Show ppErkl g PE o 6 Click in the first cell in the pl column Analysis Settings ERKI Name pl Color Range Show ppa d E 00s 7 Enter the pl for the sample protein Compass Software for Peggy User Guide page 370 Analysts Settings ERKI Name pl Color Range Show ppErkt 5 5 E 00s 8 Click in the first cell in the Color column then click the button Analysis Settings ERKI Name pl Color Range Show ppErkl 5 5 Oo orn 0 05 The color selection box will display Basic colors PETET COTITILTIII BEE TEEREE BEEER Bii ii u Custom colors EEREEE EKE EEEEE E Define Custom Colors gt Chapter 9 Charge Assay Data Analysis The color selected will be used to identify the sample protein peak in the peaks and capillaries table in the Analysis screen 9 Click a color or define a custom color and click OK The color selection will update in the table Analysis Settings ERKI Name pl Color Range Show ppErkl
240. nu The main menu contains individual menus that provide access to various software instrument and screen operations More details on menu commands can be found in Software Menus on page 7 File Edit View Instrument Window Help Instrument Status Bar The instrument status bar is used to start runs and cleaning protocols relay system status and show run progress More details on instrument control and status can be found in Chapter 7 Controlling Peggy ia NOTE The instrument status bar displays only when Compass software is connected to an instrument The status bar does not display on computer workstations used just for data analysis 2012 05 30_15 52 06_DEX70K_2mil Running e Wed 3 58 PM Thu 11 28 AM Compass Software for Peggy User Guide Software Menus page 7 Screen Tab The screen tab lets you move between Assay Run Summary or Analysis screens and is located in the upper right corner of the main window Just click a button to view a screen E Assay Run Summary g Analysis View Bar The view bar is only displayed in the Analysis screen as part of the main menu bar and allows users to switch between displaying sample chemiluminescent data fluorescent standards or capillary registration informa tion data for a single capillary or all capillaries in the run or grouped capillary data View bar options are detailed in Switching Between Sample Standards and Registration Data Views on page 146
241. o When this option is selected the following pop up box appears to let you enter a specific capillary number or range of capillaries Enter cycle and capillary descriptor Examples 2 3 1 1 12 Cycle 2 capillary 3 and cycle 1 capillaries 1 through 12 1 3 6 8 10 Cycles 1 through 3 capillaries 6 8 and 10 6 Ifyou need to change the analysis group used for a data set click the cell in the Settings column and click the down arrow Select a group from the drop down list Override Apply To Settings Hi phospho HeLa HeLa 7 Repeat the previous steps to apply other groups to specific run data 8 To remove a data set click on its cell in the Apply To column then click Remove 9 Click OK to save changes Compass Software for Peggy User Guide Images Analysis Settings page 357 Images Analysis Settings The Images analysis settings page lets you see what chemiluminescent exposures were taken during the run and view data for different exposures in the Analysis screen To access these settings select Edit in the main menu and click Analysis then click Images in the options list Analysis Training data_Hi Low ERK cbz type filter text Advanced Images Luminescence Peak Fit Peak Names Standards Exposure 240 seconds Exposure3 240 seconds Exposure3 240 seconds ne Exposure3 240 seconds r Exposure3 240 seconds hi Exposure3 240 seconds j Exposure3 240 seconds se Exposure3 240 seconds ne Exposu
242. o stack plots is not checked the colors shown in the preferences screen will be applied only to overlaid electropherograms when the Overlay the Plot option is selected in the graph pane When this option is deselected plots will use Compass default colors Click Apply to apply changes to any open run files in Compass Click Restore Defaults to restore Compass default settings Click OK to save changes and exit Click Cancel to exit without saving changes Compass Software for Peggy User Guide Selecting Custom Plot Colors for Graph Overlay page 393 Changing Plot Colors 1 Select Edit in the main menu and click Preferences then click Graph in the preferences list 2 Click the color button next to a plot number The color selection box displays 3 Select a color or define a custom color and click OK The color button will update to the new color selected 4 Repeat the steps above for any other plot colors 5 Check Apply Colors to Stacked Plots if you want the new color scheme to also be used for the Stack the Plots option in the graph pane 6 Click Apply to apply changes to plots currently displayed in the graph pane 7 Click OK to save changes and exit When the Overlay the Plots option is selected in the graph pane the new color scheme will be used Compass Software for Peggy User Guide page 394 Chapter 10 Setting Custom User Preferences Setting Peggy Up to Send Tweets Select Edit in the main menu and c
243. o use other schedule options please contact ProteinSimple Technical Support Step 6 Add Assay Plate Annotations Optional Custom reagent names notes and annotations can be entered for individual rows and wells on the assay plate This information will be stored with assay and run data During post run analysis this information will be used to apply custom analysis settings to specific sample names primary antibody names or sample and primary antibody attributes This will be explained in more detail in Compass Analysis Settings Overview on page 219 NOTE Template pane information can also be added or updated after a run is complete Compass Software for Peggy User Guide page 80 Chapter 4 Charge Assays To enter annotations 1 Click on the Template tab The default annotations for reagent rows and individual wells on the assay plate will display 2 Change or add row and well annotations as needed To do this a To enter annotations for a specific well Right click the well and select Edit or click Edit in the upper right corner of the pane or double click the selected well The following box will display Notes Attribute Compass Software for Peggy User Guide Creating a New Assay page 81 Enter the reagent name notes or specific attributes for example sample concentration or dilution ratio g Well Content Mame Hela Attribute 1 mg mL b To enter annotations for m
244. o view in the experiment pane Data for only the rows selected will display in all data views and results tables The following example shows sample data displayed in the graph in multiple view and capillaries panes when multiple rows are selected File Edit View Instrument best BiMs k Fe Assay CA Run Summary EEF Analysis El Experiment _ ee televrro N w 4 8 S Mo w 8 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 w Peak Name Position MW kDa Area re A Fe FF eww ww w Ww Compass Software for Peggy User Guide Viewing Run Data page 153 e Tolook at data for multiple sequential capillaries Hold the Shift key and select the first and last rows you want to view in the experiment pane Data for only the selected rows will display in all data views and results tables The following example shows standards data displayed in the image and peaks panes when multiple sequential rows are selected 2012 03 07_HeLa_ERK STAT3 PLCg_Day1_PLO03 Compass fo S File Edit View Instrument Window Help 5 E Hii es E Assay Run Summary EE Analysis E Experiment gt O Ge Graph 2 Image Lane m Sample Primary Cy Uses Biotin Blocki 1 Cycle 1 Samples Exp 960 0 HeLa Blocki 1 HeLa Blocki 1 HeLa Blocki 1 HeLa Blocki 1 HeLa Blocki 1 HeLa Blocki 1 HeLa Blocki 1 HeL
245. oftware Menus Active in the Assay Screen 63 REAGENT COO COGING oei eee tacos ADRESA 65 QDEMIIG OA ASSOY ea hi odie oet Cp wale itunes 66 Creating a NCW ASSQY oc iareciaerenr dananundvauerne 6 Step P ORENG TEMDIGICASSOY satnasesatuaweds 6 7 Step 2 Assign Assay Plate Reagents Optional 68 Step 3 Modifying the Assay Protocol Optional 72 Step 4 Add Assay Notes Optiondl Ti Step 5 Select a Schedule Optional 78 Step 6 Add Assay Plate Annotations Optional 79 SCD 7 SAVE MC ASS OV eaan 84 Step 8 Modify Default Analysis Parameters OO a ETE PE A O E E 85 Making Changes to an Existing ASSAY 0605 86 Switching Between Open ASSAYS 6 6 00 cece cece 86 CREatING Gd Template ASSOY 860442 ua ttiweolivera ss 8 Viewing and Changing the Detection Exposures 88 Copying Protocols and Templates 6 000 89 Copying an Assay Protocol ccc cece eee 89 CODVING GN ASSAY TEMDIGIC ci sacs Ee aa 90 PRATING FP FOTOCOIS CNG LEM IDIOTES srna 90 Pinn GNASSGY PTOTOCO srice enti ern 90 PHATING ONASSAV TEMPO seier n 90 Importing and Exporting Protocols and Templates 91 IMpPOrting GN ASSAY PrO COl s seraa R 97 EXDOMING GM ASSAY PIOlOCO orsina so RL 92 Importing an Assay TEMPla E esnin 92 Exporting an Assay Template 06 93 Chapter 5 Running a Charge Assay on Peggy 95 AO OHIO onic sien tis kl aut eit eee nas Sion 96 DLO PGCE COG akc inmate A E OR 96
246. oftware for Peggy User Guide page 198 Chapter 8 Size Assay Data Analysis Stacking Multiple Electropherograms Electropherogram data for multiple capillaries can be stacked vertically in the graph pane for comparison To do this 1 Click Single View 2 Select multiple rows in the experiment pane 3 Click the Stack the Plots button The individual electropherograms for each row selected will stack in the graph pane sgg gees 3 882 FE ak op D E a E a p E 2 E a E 0 G 2000 D E E 5 D E E ak i MW kDa Users can customize the colors used for the stacked plot display For more information see Selecting Cus tom Plot Colors for Graph Overlay on page 392 Compass Software for Peggy User Guide Changing the Electropherogram View page 199 Overlaying Multiple Electropherograms The electrooherogram data for multiple capillaries can also be overlaid on top of each other for comparison in the graph pane To do this 1 Click Single View 2 Select multiple rows in the experiment pane 3 Click the Overlay the Plots button The individual electropherograms for each row selected will overlay in the graph pane kn Graph Image Lane CE Be E Samples 1500 ppErk1 pErk1 Erk pERK2 ERK 1400 ppERK2 1300 1200 Hi phaspho HeLa C 4 Hi phospho HeLa C2 6 1100 Hi phospho HeLa C 7 1000 T o 900 T i A E 700 E 600 7 400 1 200
247. on Lets you change the pl x axis range of the data displayed Show Hidden Shows capillaries that are hidden from the data view Compass Software for Peggy User Guide page 268 Chapter 9 Charge Assay Data Analysis Opening Run Files You can open one or multiple run files at a time to compare data between runs Opening One Run File NOTE If you need to open a run file when another run is executing launch another instance of Compass first then open the file 1 Select File in the main menu and click Open Run Edit View Instrument Window Help Open Run j DemoData Add Run 2012 03 05_11 51 19_HelaControlERKassay Simple Western ERK Demo Close 2012 02 29 18 08 50 _2012Feb29_aslAb 25min Close All 2012 02 29 11 51 19 2012Feb29_aslAb 25min sue 3 ab run ee Simple Western Export Tables 2 011 08 31_16 38 23 test Export Spectra 2011 09 01 _16 41 02 Exit Browse 2 Alist of the last 10 runs opened will display Select one of these runs or click Browse to open the Runs folder and select a different file Opening Multiple Run Files NOTE Ifyou need to open a run file when another run is executing launch another instance of Compass first then open the file 1 To open the first run file select File in the main menu and click Open Run Compass Software for Peggy User Guide Opening Run Files Edit View Instrument Open Run Add Run Cl
248. onjugated antibody is aspirated Capillaries are then transferred to an incubator tray When incubation is complete Wash Buffer is aspirated Detect Step Capillaries are moved to the assay plate in the sample tray and Luminol Per oxide solution is aspirated Capillaries are then transferred to the separation tray where the emitted chemiluminescent light is detected with the CCD camera Results Step Results are available in the Analysis screen Hold Step This step is used for overlapping or overlapping with hold schedule options A cycle that has completed a specific step will go into a hold step while the remaining cycles execute the same step in parallel For example once cycle1 completes the separate step it may go into a hold step while cycle 2 executes the separate step This execute and hold pattern will continue until all cycles have completed the separate step When a run is in progress icons for steps that have not executed will be grey inactive In the following example the Secondary Antibody 2 step is executing and the Detect and Results steps have not started sample Sep Hold 1 2 Detect Results oT of 4 ar y i cry ro r pe 4 34PM 4 36PM 3 31PM 8 52PM 93 59PM 10 25PM 10 32 PM Compass Software for Peggy User Guide Watching Standards Separation Movies page 115 Watching Standards Separation Movies Users can view a movie of the fluorescent standards separation in all 12 capilla
249. or click Edit in the upper right corner of the pane or double click the selected well The following box will display Enter the reagent name notes or specific attributes for example sample concentration or dilution ratio Compass Software for Peggy User Guide Creating a New Assay page 39 Click OK The new information will display in the selected well b To enter annotations for multiple wells or a row To select individual wells press and hold the Control key and then select individual wells To select a sequential set of wells or a full row press and hold the Shift key then select the first well and last well Next right click and select Edit or click Edit in the upper right corner of the pane The following box will display Stacking Matrix Compass Software for Peggy User Guide page 40 Chapter 2 Size Assays Enter the reagent name notes and specific attributes for example sample concentration or dilution ratio NOTE When annotations are being edited an asterisk will appear in the Template tab to indicate changes have been made and should be saved Only Name and Attribute will be used by Compass to annotate the data Compass Software for Peggy User Guide Creating a New Assay page 41 Step 7 Save the Assay 1 Select File from the main menu and click Save As Enter the assay name and click Save JE documents My Docum
250. ort Template The following window displays Organize v New folder wr Favorites Documents library E Desktop Assays ie Downloads S Recent Places Arrange by Folder v Name Date modified Type __ Standard sample template template 10 4 2011 10 10 TEMPLATE File MB Desktop Libraries E Documents iB My Documents Compass vE m File name New template v Template File template y 3 The default directory will be Compass Assays Change the directory if needed 4 Enter a template name and click Save The template will be saved as a template file Compass Software for Peggy User Guide page 51 Chapter 3 RUNNING a Size Assay on Peggy Chapter Overview Starting a Run Stopping a Run Compass Software for Peggy User Guide page 52 Chapter 3 Running a Size Assay on Peggy Starting a Run Step 1 Get Ready il 2 By 4 6 Open Compass software Prepare instrument empty waste refill water and add a new manifold sponge Create or open desired assay file Prepare assay plate following the procedure described in the product insert IMPORTANT To prevent well evaporation and ensure best results keep a lid on the assay plate until ready to use While plate is spinning add Wash Running and Matrix Removal Buffers to resource tray cups Place cap illary box in the designated resource tray position IMPORTANT Capillaries are light sensitive Keep the cover on the box until you are rea
251. ose Close All Save Save As Export Tables Export Spectra Exit Window page 269 Help DemoData 2012 03 05_11 51 19_HelaControlERKassay Simple Western ERE Demo 2012 02 29 18 08 50_2012Feb29_aslAb 25min 2012 02 29 11 51 19 2012Feb29_aslAb 25min 3 ab run Simple Western 2011 08 31_16 38 23 test 2011 09 01_16 41 02 Browse 2 Alist of the last 10 runs opened will display Select one of these runs or click Browse to open the Runs folder and select a different file 3 To open another run file select File in the main menu and click Add Run Open Run Add Run Close Close All Save Save As Export Tables Export Spectra Exit File Edit View Instrument Window Help 2011 08 31_16 38 23 test 3 ab run 2011 09 01_16 41 02 Browse 4 Alist of the last 10 runs opened will display Select one of these runs or click Browse to open the Runs folder and select a different file When a run is added its data will append to the open run file and will display as a second set of up to 96 capillaries in all screen panes The second run file name will also appear in the Compass title bar Compass Software for Peggy User Guide page 270 Chapter 9 Charge Assay Data Analysis 2012 03 05_11 51 19 HelaControlERKassay 2011 08 31_16 38 23_test Compass File Edit View Instrument Window Help eet EiHe Chemiluminescence ey Wce ee Soe a eee eee oe S60 5
252. osition MW kDa Height Area Area Data for the row s selected in the experiment pane will display as follows Electropherograms in the graph pane display overlaid or stacked depending on the graph option chosen e Results for only the selected row s will display in the peaks and capillaries tables The selected row s are highlighted in the image pane Compass Software for Peggy User Guide Viewing Run Data page 157 his Graph 2 Image Lane Cycle 1 Samples Exp 30 0 Mm Lanes for only the selected row s are displayed in the lane pane Ke Graph 5 Image 5 Lane PMB DA Compass Software for Peggy User Guide page 158 Chapter 8 Size Assay Data Analysis To view data up to 96 capillary format Click Multiple View in the View bar or select View in the main menu and click Multiple View Edit mw i fg E Assay TE Run Summary Sample Primary Biotin Blocki HeLa Blocki HeLa Blocki HeLa Blocki HeLa Blocki Hela Blocki HeLa Blocki 1 HeLa Blocki 1 HeLa Blocki 1 HeLa Blocki Hela Blocki Biotin Blocki HeLa Blocki HeLa Blocki HeLa Blocki HeLa Blocki HeLa Blocki Blocki HeLa Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki
253. ove Window Peas sila Override Peak Find Settings Threshold Width Restore Original Click Import to import an analysis settings file This will be explained in more detail in Importing Analysis Settings on page 386 Click Export to export the current analysis settings file This will be explained in more detail in Exporting Analysis Settings on page 387 Click Restore Original to restore Compass default settings e Click OK to save changes and exit Click Cancel to exit without saving changes Compass Software for Peggy User Guide Peak Fit Analysis Settings page 361 Range Settings Minimum The pl value below which peaks will not be identified This value will also be used as the default lower pl range for the data displayed in the electropherogram and virtual blot The default value is 5 Maximum The pl value above which peaks will not be identified This value will also be used as the default upper pl range for the data displayed in the electropherogram and virtual blot The default value is 7 Baseline Settings Threshold The variance or roughness in a baseline data segment below which a point will be called part of the baseline The default value is 1 0 Window How long baseline data segments are expected to be in pixels Shorter segments will allow the baseline to follow plateau sections of the signal The default value is 15 Stiffness The amount the baseline is allowed to vary f
254. p t Separation Matrix b Stacking Matrix gt Sample Separation Time min 40 0 40 0 40 0 40 0 40 0 40 0 40 0 40 0 t Separation Voltage volts 250 250 250 250 250 250 250 250 gt Matrix Removal t Blocking Time min 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 4 Primary Ab Time min 120 0 120 0 120 0 120 0 120 0 120 0 120 0 120 0 Well Row ee a Washes 2 2 2 2 2 2 2 2 Wash Soak Time sec 150 0 150 0 150 0 150 0 150 0 150 0 150 0 150 0 4 Secondary Ab Time min 60 0 60 0 60 0 60 0 60 0 60 0 60 0 Well Row rr o a o a a a Washes 2 2 2 2 2 2 2 2 Wash Soak Time sec 150 0 150 0 150 0 150 0 150 0 150 0 150 0 150 0 t Detection 6 If needed change the secondary incubation reagent row location To do this click the cell in the value column next to Well Row and select a different row on the assay plate Protocal b E Notes Add Remove Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle amp Separation Matrix Stacking Matrix Sample Separation Time min 40 0 40 0 40 0 40 0 40 0 40 0 40 0 40 0 Separation Voltage volts 250 250 250 250 250 250 250 250 Matrix Removal Blocking Time min 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 Primary Ab Time min 120 0 120 0 120 0 120 0 120 0 120 0 120 0 120 0 Secondary Ab Time min 60 0 60 0 60 0 60 0 60 0 60 0 60 0 60 0 Well Row D1 z rr o o o o a Washes D1 2 2 2 2 2 2 2 Wash Soak Time sec 150 0 150 0 150 0 150 0 150 0 150 0 150 0 Detection NOTE Only rows designa
255. p Plots m soon aert Primary Capillary Primary 1 Primary 2 Primary 1 Primary 2 ERKI Std Dev CV PLCg The mean values for named peaks in each group are plotted in bar graphs with error bars showing the stan dard deviation The plots compare different antibodies for the same sample and different samples for the same antibody to allow a choice of presentation Compass Software for Peggy User Guide Group Statistics page 179 233 Peak Groups 3 Capillary Groups HH SampleA Run 1 SampleB Run 1 n I Average area Average area Average area Average area Hiding or Removing Capillaries in Group Analysis Hidden capillaries are not included in groups However hiding capillaries provides an easy way to reject indi vidual capillaries from the statistical analysis See Hiding Capillary Data on page 159 for details on how to do this Larger groups of capillaries can also be excluded from analysis To do this select View and click Filter Compass Software for Peggy User Guide page 180 Chapter 8 Size Assay Data Analysis Capillaries os Oi e Wo mh of oo oh in e lu u e a So O0 0O 0O R ARARA RAOK Ba El Show named peaks only Ce Uncheck the box next to the cycles or capillaries you wish to remove and click OK This data will now be removed from the grouped view statistics Compass Software for Peggy User Guide Copying Data Views and Results Tables page 181
256. page 44 Chapter 2 Size Assays Creating a Template Assay Users can create and save template assays that can be used as a starting point for creating new assays To do this 1 Select File in the main menu Click Open Assay to open an existing assay or click New Assay to open an existing template assay 2 Follow the steps in Creating a New Assay on page 25 to make changes to the assay 3 When changes are complete select File in the main menu and click Save As Select the New Assays folder Save As Go Organize v New folder a Favorites BB Desktop m3 Downloads S Recent Places Documents library New Assays Name Arrange by Folder Date modified Type Simple Western assay 9 26 2011 3 25PM Compass Assay File 4 WW Desktop 4 lt j Libraries a E Documents s E My Documents a d Compass Ji Assays d New Assays d Runs m File name Protein Test ins Save as type Assay File assay z Hide Folders 4 Type the name for the new template assay and click Save 5 Select File in the main menu and click New Assay The new template assay will now be available in the drop down list File Edit Instrument Window Help Protein Test Simple Western Compass Software for Peggy User Guide Viewing and Changing the Detection Exposures page 45 Viewing and Changing the Detection Exposures To view the current detection profile roll the cursor over the ex
257. pass Software for Peggy User Guide page 282 Chapter 9 Charge Assay Data Analysis e Data in this view shows capillary registration only e Graph view data displays electropherograms in fluorescence y axis and position x axis Lane view data displays capillary registration only Image view data displays capillary registration only e Registrations are highlighted in both the peaks and capillaries tables and marked with an R Because capillaries must be moved multiple times during the run their positions are tracked to account for potential changes in imaging position as they are moved to and from the separation block To do this one of the standards is used as a capillary registration An image of the standard used for registra tion is taken at the end of the separation step prior to chemiluminescent imaging The position of this peak in the registration image is then compared to the position of the same standard peak in the stan dards image The shift value listed in the peaks table is the change in pixel position between the two images Compass data is then aligned to account for any changes in capillary position For information on checking and identifying registration peaks see Step 3 Checking Capillary Regis trations on page 299 Compass Software for Peggy User Guide Viewing Run Data page 283 Selecting and Displaying Capillary Data You can choose to view data from one multiple or all capillaries at once
258. pen ata time Lower Incubator To close a tray click the corresponding tray button again NOTE If the tray contro window is closed when a tray is open the tray will close automatically Cleaning Two cleaning options are available for Peggy Manual Clean This option is used for general manual cleaning and cleaning the manifold head To do a manual cleaning select Instrument and click Manual Clean The manifold head will move to a safe position for easy access and the vacuum will turn on NOTE Please contact Protein Simple Technical Support if you have any questions regarding the manifold cleaning procedure Cleanup This option is a fully automated cleaning step The manifold head is flushed the separation tray troughs are aspirated and washed and any capillaries left in the trays or gripper are picked up and discarded This option should be selected when the instrument has not been used for more than a week or if a run error occurs Cleaning takes about eight minutes to complete Compass Software for Peggy User Guide page 122 Chapter 7 Controlling Peggy To start the protocol select Instrument and click Cleanup A window will appear with instructions Cleanup Make sure the separation tray troughs are clean before clicking OK To clean the troughs click Cancel and use the Open Trays menu to access the separation tray troughs Cleaning the troughs may require soaking them with distilled water F the
259. perature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 36 250 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 41 267 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 46 282 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 51 298 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 21 56 313 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 22 01 329 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 22 06 344 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 22 11 360 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 22 16 377 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 22 21 394 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 22 26 412 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 22 31 429 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 99 LookupPWM 8 2011 09 25 10 22 36 446 temperature INFO SetPoint C 23 0 Chamber C 24 99 Ambient C 24 9
260. pherograms in fluorescence y axis and position x axis Lane view data displays fluorescent standards only Image view data displays fluorescent standards only Standards are highlighted in both the peaks and capillaries tables and marked with an S For information on checking and identifying standards peaks see Step 2 Checking Fluorescent Sizing Standards on page 296 To view registration data Click Show Registrations in the View bar or select View in the main menu and click Registration DemoData_Charge Hela ERK12 Compass Lo k File Edit View Instrument Window Help Assay o Run Summary Sample High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 A an ae aut pura Neen N ma Graph k Image Lane tel Orro Registrations Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 Fluorescence o NW Se OH I OO Reg 49 500 Position Low phospho HeLa in 5 8 C1 4 Reg 7 3 fe Peaks E Capillaries Primary ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 FRK1 Cap Peak Position C14 c1 4 C1 4 C1 4 C1 4 C1 4 Ci 4 c1 4 1 268 282 311 339 351 360 874 900 Height 15 11 179 07 0 6 0 5 2 2 2 8 Com
261. pho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 W 35 Peaks N F Capillaries Sample Primary Cap Peak Name Position pl Height Area Area Width S N Compass Software for Peggy User Guide page 286 Chapter 9 Charge Assay Data Analysis To look at data for all capillaries Hold the Shift key and select the first and last rows in the experi ment pane Data for all rows will display in all data views and results tables The following example shows sample data displayed in the lane and peaks panes when all rows are selected The pane scroll bars can be used to view all 8 cycles e DemoData_Charge_Hela_ERK12 Compass Ee File Edit View Instrument Window Help B E l fg E Assay PE Run Summary E E Sample High phos High phos High phos High phos High phos High phos High phos High phos High phos High phos High phos High phos High phos High phos High phos High phos r aligns Low phosp Low phosp Low phosp Low phosp Low phosp Low phosp Low phosp Low phosp Low phosp Low phosp Low phosp Low phosp Low phosp Low phosp Low phosp Low phosp cies ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2 ERK1 2
262. plays Compass release notes for the current and prior versions Compass Log Displays the Compass software log file About Compass Displays the Compass software version and build information Changing the Compass Main Window Layout The Compass main window can easily be resized as can the individual panes in each screen Screen panes can also be moved outside of the main window Resizing the Main Compass Window To resize the main window roll the mouse over a corner or border until the sizing arrow appears Then just click and drag to resize Resizing the Screen Tab The screen tab can be sized to show all or just some of the screen buttons To resize roll the mouse over the left edge of the tab until the sizing arrow appears then click and drag to resize If a screen button is hidden a double arrow will display in the tab Click to display and select the hidden screen kid Assay p Run Summary Compass Software for Peggy User Guide Changing the Compass Main Window Layout page 11 Resizing Screen Panes To resize a pane Roll the mouse over the pane border until the sizing arrow appears Then just click and drag to resize To maximize a pane Click the maximize button in the upper right corner or double click the tab The other panes in the screen will automatically minimize to pane bars in the task area along the win dow border File Edit View Instrument Window Help u E e E pssry R
263. plied to the run data Modifying a Peak Fit Group 1 Select Edit in the main menu and click Analysis then click Peak Fit in the options list 2 Click on the group in the analysis settings box you want to modify Compass Software for Peggy User Guide page 364 Chapter 9 Charge Assay Data Analysis Analysis Settings Peak Fit STAT peak fit 3 Modify range baseline or peak find parameters as needed 4 Click OK to save changes The new peak fit settings will be applied to the run data Deleting a Peak Fit Group 1 Select Edit in the main menu and click Analysis then click Peak Fit in the options list 2 Click on the group in the analysis settings box you want to delete and click Remove Analysis Settings Peak Fit STAT peak fit 3 Click OK to save changes Applying Peak Fit Groups to Specific Run Data 1 Select Edit in the main menu and click Analysis then click Peak Fit in the options list 2 Click on the group in the analysis settings box you want to apply to specific run data Analysis Settings Peak Fit STAT peak fit 3 Application of peak fit groups to specific run data is done in the override box Click Add under the over ride box A default override data set will be created from sample information found in the run file Compass Software for Peggy User Guide Peak Fit Analysis Settings page 365 Override Apply To Settings Hi phospho HeLa STAT peak fit Add Remove 4 Click t
264. plying Analysis Groups to Specific Run Data 1 Select Edit in the main menu and click Analysis then click Advanced in the options list 2 Click on the group in the analysis settings box you want to apply to specific run data Compass Software for Peggy User Guide page 226 Chapter 8 Size Assay Data Analysis Analysis Settings Advanced STAT analysis 3 Application of analysis groups to specific run data is done in the override box Click Add under the over ride box A default override data set will be created from sample information found in the run file Override Apply To Settings Biotinylated Ladder STAT analysis Add Remove 4 Click the cell in the Apply To column then click the down arrow Override Apply To Settings biotin ladder Advanced 2 biotin ladder Custom Settings 5 Select an option from the drop down list This will apply the settings group selected to specific run data as follows Sample names All sample names entered in the assay template Assay screen will display in the drop down list otherwise the default name of Sample will be shown Select a sample name to apply group settings to all capillaries that use this sample name in the run file Attributes All sample attributes entered in the assay template Assay screen will display in the drop down list Select an attribute to apply group settings to all capillaries that use this attribute in the run file Compass
265. posures cell in Protocol pane gt Separation Matrix t Stacking Matrix gt Sample Separation Time min 40 0 t Separation Voltage volts 250 bt Matrix Removal t Blocking Time min 15 0 Primary Ab Time min 120 0 t Secondary Ab Time min 60 0 4 Detection Well Row 6 Detection Profile 6 Exposures signal 30 0 signal 60 0 se signal 960 0 se While ProteinSimple recommends using the default assay detection profile users can change the profile exposure times and exposure sequences if needed To do this 1 Select the exposures cell in the Protocol pane and click the button or double click in the cell The fol lowing screen will display R m bb Exposure sec 30 0 60 0 120 0 240 0 480 0 960 0 Compass Software for Peggy User Guide page 46 Chapter 2 Size Assays Each row represents an individual exposure that will be taken during the run a To change an existing exposure time Click in the exposure cell and enter a new time in seconds Detection Profile Exposure sec 30 0 60 0 120 0 240 0 480 0 b To delete an existing exposure Select a type or exposure cell and click Remove c To add anew exposure Select Add A new exposure will be added to the end of the list Click in the exposure cell and enter an exposure time in seconds 2 Click OK to save and exit Copying Protocols and Templates The steps and parameters in the Protocol
266. proteinsimple Compass Software for Peggy User Guide Copyright 2013 ProteinSimple All rights reserved ProteinSimple 3040 Oakmead Village Drive Santa Clara CA 95051 Toll free 888 607 9692 Tel 408 510 5500 Fax 408 510 5599 email support proteinsimple com web www proteinsimple com Compass Software for Peggy User Guide P N 041 817 Revision 2 June 2013 For research use only Not for use in diagnostic procedures Patents and Trademarks Automatic Image Capture AIC and digital ProteomeChip technology are covered by U S Patent Nos 6 995 901 6 909 459 6 853 454 6 271 042 7 166 202 and other issued and pending patents in the U S and other countries ProteinSimple the ProteinSimple logo the Aloha Innotech logo Protein Forest the Protein Forest logo the AIC logo AlphaCal Alohalmager AlohaPart11View AlohaQuant AlohaSnap AlohaSpec AlphaUV AlohaView ChemiGlow Chromalight dPC digital ProteomeChip FluorCchem MSRAT Multilmage NanoPro the intertwined helix design iWB Peggy ProteomeChip red Sally Simon Simple Western Spectra Plex Xpedition XplorBright and XplorUV are trademarks or registered trademarks of ProteinSimple Other marks appearing in these materials are marks of their respective owners Table of Contents Chapter 1 Getting Started with Compass 1 EQUNCHING TNC SONWOlC xii ee ss aii dteon tones 2 SOW GI EOV Werra E 2 CAGNGING MESaTeen VICW geert irern titans 3
267. r Peggy User Guide Result PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED PASSED Select a log file and click View The individual test details will display Failure Reason Save File As page 129 page 130 Chapter 7 Controlling Peggy Peggy s Status Modes The instrument status bar displays status buttons and progress bars depending on what Peggy is doing Ready New Run button Peggy is ready but an assay is not loaded Click New Run to open an assay Ready Start button Peggy is ready and an assay is loaded Click Start to begin a run Not Ready Clean button Peggy is not ready and must perform system cleaning Click Clean to start the cleaning protocol Not Ready Reset button Peggy is not ready and must reinitialize Click Reset to start the initializa tion protocol Running Stop button Peggy is running an assay The run name time the run started and when it will complete display in the run progress bar Click Stop to stop the run Cleaning button not active Peggy is running a cleaning protocol The time the cleaning protocol Started and when it will complete display in the run progress bar Error Reset button An error has occurred Go to the Status window in the Run Summary screen to view details When the source of the error is corrected click Reset Compass Software for Peggy User Guide page 1
268. r Sample Data If Compass did not automatically name a sample protein associated with a specific primary antibody this can be adjusted manually To do this 1 Click Show Samples in the View bar 2 Click Single View in the View bar 3 Click on the row in the experiment pane that contains the sample you wish to correct then click the Graph tab Compass Software for Peggy User Guide Changing Sample Protein Identification page 187 4 Right click the peak in the electrooherogram or peaks table and click Name then click a name in the list Compass will change the peak name in the electropherogram and results tables and adjust peak names for other sample proteins accordingly Tak T ity o wa ary E E wg a Zoom Out Remove Peak Hide Mame Add Peak Add Baseline Point Remove Baseline Point Clear All Primary Cap Peak Name Position MW kDa K562 antiEn QS 1 RR SQL B K562 0 anti B NOTES For details on how to specify peak name settings see Peak Names Settings on page 238 Virtual blot data in the lane pane will also update to reflect changes made in the graph pane Compass Software for Peggy User Guide page 188 Chapter 8 Size Assay Data Analysis Displaying Sample Data for Named Peaks Only You can adjust the sample data to display results only for user specified named peaks To do this 1 Click Show Samples in the View bar 2 Click View in the main menu an
269. r all capillaries at once e Tolook at data for one capillary Click a row in the experiment pane Data for just the row selected will display in all data views and tables The following example shows sample data displayed in the lane and peaks panes when one sample is selected a ioe Ex File Edit View Instrument Window Help ES Ms tg E Assay Pg Run Summary EE Analysis TEbxperiment h E Graph E Image E Lane _ H els 5 Primary Cyc B hela Blocki 1 a hela Blocki ia hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki oe NINN NNN NNN NNN FR e Cap Peak Name Position MW kDa Ie amp F amp F F amp F Fe Fe FFF FF FW WwWHw ssw w Compass Software for Peggy User Guide page 152 Chapter 8 Size Assay Data Analysis e Tolook at data for multiple non sequential capillaries Hold the Ctrl key and select the rows you want t
270. re 240 seconds a Restore Original Click Import to import an analysis settings file This will be explained in more detail in Importing Analysis Settings on page 386 Click Export to export the current analysis settings file This will be explained in more detail in Exporting Analysis Settings on page 387 Click Restore Original to restore Compass default settings Click OK to save changes and exit Click Cancel to exit without saving changes Compass Software for Peggy User Guide page 358 Chapter 9 Charge Assay Data Analysis Exposure Settings The exposure used for the sample data displayed in the Analysis screen is shown in the All box Cycle Luminescence All l 240 seconds 1 240 seconds 2 240 seconds 3 240 seconds 4 240 seconds J 240 seconds 6 240 seconds T 40 seconds B 240 seconds NOTE Peggy runs up to eight cycles 12 capillaries per cycle The individual exposure setting selected for a specific cycle is applied for all 12 capillaries in that cycle unless selected for All cycles Exposure Sample data displayed in the Analysis screen is for this specific exposure only To see the number of exposures and exposure times used for the run data click the arrow in the drop down list next to All Compass Software for Peggy User Guide Images Analysis Settings page 359 Cycle Luminescence All Exposure3 240 seconds 2 3 Exposure 240 seconds
271. redicted molecular weights based on sample and assay conditions 12 Select the checkbox in the first cell of the Show column This will turn peak naming on for the sample protein Ana bys is Setti ngs ERKL Mame MW Color Range Show ERKO n E 10 To turn peak naming off for a particular sample protein deselect the checkbox in the Show column 13 To add another sample protein click Add under the analysis settings peak table Analysis Settings ERK1 2 Color Range Show fe 0 Compass Software for Peggy User Guide Peak Names Settings page 243 14 Repeat the previous steps to enter information for other sample proteins In the following example two Sample proteins were entered Analysts Settings EREL 2 Color Range Show E To remove a sample protein select its row and click Remove 15 Click OK to save changes Adding Peak Names Groups 1 Select Edit in the main menu and click Analysis then click Peak Names in the options list 2 Click Add under the analysis settings box A new group will be created Analysis Settings ERRL 2 ERRL 2 2 3 Click on the new group and enter a new name Compass Software for Peggy User Guide page 244 Chapter 8 Size Assay Data Analysis Analysis Settings 4 Enter information in the analysis settings peak table as described in Creating a Peak Names Group on page 239 5 Click OK to save changes Modifying a Peak Names Group
272. ries To do this 1 Click the Separation tab Separation IV Plot E Cycle 1 P a0 op FB 2 The player control panel has play pause rewind and fast forward buttons and a slider bar that allows you to scroll through the movie manually P 40 OP HE Compass Software for Peggy User Guide page 116 Chapter 6 Run Status Click Play button on far left to view the movie In the examples below standards for a size assay are on the left and standards for a charge assay are on the right i iii nA a a See ee NOTE Complete separation movies of the fluorescent standards are not available until the separation step has finished executing If the movie is played while the separation step is executing the movie will only show separation progress up to the current point in time Compass Software for Peggy User Guide Viewing Current and Voltage Plots page 117 Viewing Current and Voltage Plots Users can view plots of the total current and voltage measured during separation for all 12 capillaries To do this click the IV Plot tab In the examples below the IV plot for a size assay is on the left and the IV plot for a charge assay is on the right Separation 5 IV Plot 76 Separation IV Plot PO Zoom Out Zoom Out Cycle 1 Cycle 1 310 1900 65 0 300 160 1800 t 62 5 60 0 57 5 t 55 0 52 5 50 0 147 5 t 45 0 42 5 150 1700 140 1600 130 1500 1400 120 13004 i 1200 100 11004
273. rom a straight line Settings between 0 1 and 1 0 make the baseline fit closer to a straight line Settings from 1 0 to 10 0 will make the baseline fit fol low the data more closely The default value is 1 0 Peak Find Settings Threshold The minimum signal to noise ratio required for a peak to be identified A setting of 1 0 will detect many peaks a setting of 30 0 will detect fewer peaks The default value is 20 0 Width The approximate peak width at full width half max in pixels used to detect peaks The mini mum value for this setting is 3 0 Larger widths help to eliminate the detection of shoulder peaks and noise peaks The default value is 7 0 Peak Fit Analysis Settings Groups Peak fit settings are saved as a group and multiple settings groups can be created Specific group settings can then be applied to individual capillaries sample names or attributes in the run data NOTES ProteinSimple recommends using the Compass default values for peak fit analysis settings These settings are included in the default Peak Fit group Analysis settings are run file specific However settings can be imported or exported for use with other run files For more information see Importing and Exporting Analysis Settings on page 386 Compass Software for Peggy User Guide page 362 Chapter 9 Charge Assay Data Analysis Peak fit groups are displayed in the analysis settings box Analysis Settings Add Remove The Peak Fit gro
274. rotocol protocol Arrange by Folder Date modified Type 10 4 2011 9 57 PM PROTOCOL File mW r File name New protol ye Save as type Protocol File protocol z a Hide Folders 3 The default directory is Compass Assays Change the directory if needed 4 Enter a protocol name and click Save The protocol will be saved as a protocol file Importing an Assay Template NOTE Importing an assay template imports information into the Template pane only Open the assay you want to import the assay template in to Select File in the main menu and click Import Template Select a template file template and click OK The imported information will display in the Template pane Compass Software for Peggy User Guide Importing and Exporting Protocols and Templates page 93 Exporting an Assay Template NOTE Exporting an assay template exports information in the Template pane only 1 Open the assay you want to export the assay template from 2 Select File in the main menu and click Export Template The following window displays Organize v New folder wr Favorites Documents library BD Desktop Assays m3 Downloads S Recent Places Arrange by Folder Name Date modified Type _ Standard sample template template 10 4 2011 10 10 TEMPLATE File MB Desktop Libraries E Documents iB My Documents c File name New template 3 The default directory will be Compass Assays
275. roup 1 Select Edit in the main menu and click Analysis then click Standards in the options list 2 Click on the group in the analysis settings box you want to delete and click Remove Analysis Settings Standards New Standards 3 Click OK to save changes Applying Analysis Groups to Specific Run Data 1 Select Edit in the main menu and click Analysis then click Standards in the options list 2 Click on the group in the analysis settings box you want to apply to specific run data Analysis Settings Standards New Standards Compass Software for Peggy User Guide Standards Settings page 385 3 Application of analysis groups to specific run data is done in the override box Click Add under the over ride box A default override data set will be created from sample information found in the run file Override Apply To Settings Hi phospho HeLa Mew Standards Add Remove 4 Click the cell in the Apply To column then click the down arrow Override Apply To Settings i phospho HeLa New Standards Low phosphe HeLa Cyclel Custom Settings 5 Select an option from the drop down list This will apply the settings group selected to specific run data as follows Sample names All sample names entered in the assay template Assay screen will display in the drop down list otherwise the default name of Sample will be shown Select a name to apply group settings to all capillaries that use this
276. s Assay Screen Overview The Assay screen is used to create view and edit assays To access this screen click Assay in the screen tab EA Assay chy Run Summary g Analysis Assay Screen Panes The Assay screen has four panes e Layout Displays a map of the assay plate and shows where assay reagents will be located e Protocol Lists individual assay protocol steps and parameters that Peggy will execute for each of the 12 capillaries simultaneously Notes Lets you enter specific assay information that is saved with the assay and can be used for future reference e Template Enter annotations for the individual well and row reagents in the assay plate __ Charge Compass j File Edit Instrument Window Help C3 Run Summary ik Analysis Bese Run Charge EEE Add v Remove wie xOsi1i23485L1TH5 Cyclel Separation Immobilization Time sec Primary Ab Time min Secondary Ab Time min Tertiary Ab Time min Detection VOSErAc TOMmooGy VOZErAC TOAMmMoOOGyD Compass Software for Peggy User Guide Assay Screen Overview page 63 Software Menus Active in the Assay Screen The following software menus are available File Edit Instrument when Compass is connected to Peggy Window Help The File and Edit menu options specific to the Assay screen are described next File Menu The following File menu options are active Edit Instrument Winda New Assay d
277. s The path can also be copied into the Windows Explorer address bar to launch Com pass and open the run file automatically Assay Steps Size based Assays Each assay step that is executed during the run is represented by an icon The time the individual step is scheduled to start is shown under each icon Details on what happens during each assay step is as follows Description Sample Loading Step Capillaries are moved to the assay plate in the sample tray where Separation Matrix Stacking Matrix biotinylated ladder and samples are aspirated Capillar ies are then transferred to the separation tray Separation Step Samples and fluorescent standards are separated in the capillaries Cap illaries are then exposed to UV light which immobilizes the separated proteins to the walls of each capillary During the separation voltage and current are monitored and displayed in the IV Plot Separation of the fluorescent standards is recorded and a movie of the sepa ration is compiled for viewing when separation is complete in the Separation pane After separation Matrix Removal Buffer is aspirated Blocking Step Capillaries are moved to the assay plate in the sample tray and blocking reagent Antibody Diluent is aspirated Capillaries are then transferred to an incubator tray When incubation is complete Wash Buffer is aspirated Primary Antibody 1 Step Capillaries are moved to the assay plate in the sample tray and primary antibody
278. s be C1 C8 depending on the number of cycles programmed Attributes Attribute text If attribute information was entered for any of the rows or wells in the assay template Assay screen up to four can be selected and displayed as lane labels Viewing the Uncorrected Sample Baseline 1 Click the Remove Baseline button active for sample data only This will remove the automatic base line correction Compass Software for Peggy User Guide page 322 Chapter 9 Charge Assay Data Analysis UE Graph 1E Image E Lane cep Da he a Wie thts fs we nm j 2 Click the Remove Baseline button again to return to the default baseline corrected view Overlaying Standards Data on Sample Lanes Data for the standards can be overlaid on the sample data in the virtual blot so users can view the raw unaligned uncorrected lane data To do this 1 Click the Overlay Standards Data button active for sample data only An overlay of the raw sample and standards data will display Compass Software for Peggy User Guide Changing the Virtual Blot View page 323 ie Graph 3 Image E Lane J ei BF 2N m one Standards Data MMibtdddd ga if WME Au KOSS ESO PS S S a S S a a a S a S a Se OO ee A i C0 Sa AA e aaa E T E s c SS SS 1 m m a n a lt COGO a l a Cc lt C EJ i _ cne SS _ p
279. s option is selected automatically when Baseline Fit is selected in graph options Low phospho HeLa C1 10 a TI D o wi a E E 3 a ao 950 900 850 800 750 700 650 600 500 450 SE ee a a PCP SSPE S TETE ETTE ee E EE ee 5350 5375 5 400 5 425 5450 5 475 5 500 5 525 5 550 5 575 5 600 5 625 5 650 5 675 5 700 5 725 5 750 5 775 pl Compass Software for Peggy User Guide Changing the Electropherogram View page 343 e Fit Clicking this option will display the bounding envelope of the fitted peaks as calculated by the software for the raw sample data In the example that follows both Fit and Sample are selected ppErk1 pErk1 Erk ppERK J Low phospho HeLa C1 10 a TI z T ie E a E i E T l S Compass Software for Peggy User Guide page 344 Chapter 9 Charge Assay Data Analysis Fit Baseline Corrected Clicking this option will display the fitted peaks as calculated by the soft ware for the sample baseline corrected data In the example that follows both Fit Baseline Corrected and Sample are selected the fit plot is on the bottom is al pErk1 Erki pERK2 ppERK 2 Hi phospho HeLa C13 nce D TI a E E T 1100 1000 500 200 700 600 500 400 300 200 SE 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 pl NOTE When viewing standards or registration data this option is called Standards Fluorescence or Regis tration Fluorescence
280. s will change the peak name in the electropherogram and results tables and adjust peak names for other sample proteins accordingly 8 S TI T TI a E E D 4 1000 300 200 700 4 600 500 4 400 300 4 700 4 SE 50 51 52 53 54 55 56 57 58 58 NOTES Low phaspho HeLa C1 4 Zoom Out Remove Peak Hide Name Add Peak Add Baseline Point Remove Baseline Point Clear All Copy Ctrl C For details on how to specify peak name settings see Peak Names Settings on page 367 Virtual blot data in the lane pane will also update to reflect changes made in the graph pane Compass Software for Peggy User Guide Changing Sample Protein Identification page 317 Displaying Sample Data for Named Peaks Only You can adjust the sample data to display results only for user specified named peaks To do this 1 Click Show Samples in the View bar 2 Click View in the main menu and click Filter 3 Check the Show Named Peaks only box and click OK oS Filter Capillaries cow mH i E Wh fe 209 coo Om Un e WwW oh Fe SIE SE SSE SSS SSS 88 Show named peaks only Compass Software for Peggy User Guide page 318 Chapter 9 Charge Assay Data Analysis Compass will hide peak data in the electropherogram and results tables for all unnamed peaks and instead only display data for named peaks in the electropherogram and results tables te 68 7a Samples SsaggasgE C
281. sample name in the run file Attributes All sample attributes entered in the assay template Assay screen will display in the drop down list Select an attribute to apply group settings to all capillaries that use this attribute in the run file Cycle 1 8 When this option is selected group settings will be applied to all capillaries in the cycle Custom settings Lets you choose specific capillaries to apply the group settings to When this option is selected the following pop up box appears to let you enter a specific capillary number or range of capillaries Compass Software for Peggy User Guide page 386 Chapter 9 Charge Assay Data Analysis wows Enter cycle and capillary descriptor Examples 2 3 1 1 12 Cycle 2 capillary 3 and cycle 1 capillaries 1 through 12 1 3 6 8 10 Cycles 1 through 3 capillaries 6 8 and 10 Cancel 6 Ifyou need to change the analysis group used for a data set click the cell in the Settings column and click the down arrow Select a group from the drop down list Override Apply To Settings Hi phospho HeLa Mew Standards Standards New Standards 7 Repeat the previous steps to apply other groups to specific run data 8 To remove a data set click on its cell in the Apply To column then click Remove 9 Click OK to save changes Importing and Exporting Analysis Settings The analysis settings in a run file can be exported as a separate file This allows the same anal
282. says Add v Remove ws XOsS5123485LT5 Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle 8 Separation Matrix Stacking Matrix Sample Separation Time min Separation Voltage volts Matrix Removal Blocking Time min Primary Ab Time min Secondary Ab Time min Detection wOzZErxAc TOAMmMoOOOD Step 2 Assign Assay Plate Reagents Optional 1 Click on the Layout tab This pane displays the default row locations of where each reagent should be placed on the assay plate using the Plate Guide provided with Peggy NOTE Up to 96 different samples or conditions lysates proteins antibodies or incubation times can be assayed using the full eight cycles to accommodate experimental needs Compass Software for Peggy User Guide Creating a New Assay page 27 Row A Sample and Biotinylated Ladder TOFECZ tTranmooap E L M KAXAK ENEN N f Separation Matrix 0 sacking Matix P Row B Blocking Antibody Diluent Row C Antibody Diluent C1 and Primary antibody C2 C12 Row D Streptavidin HRP D1 and Secondary HRP conjugate D2 D1 2 Row J Luminol S Peroxide mix Row M Water M5 M20 Row N Water N5 N6 and N19 N20 and Separation Matrix N7 N18 Row O Water 05 06 and 019 020 and Stacking Matrix 07 018 Row P Water P5 P20 NOTE For details on sample reagent and assay plate preparation please refer to the product insert pro vided with the Simple Western kits
283. scence 3004 200 100 000 200 200 700 600 200 400 300 200 gS E Compass Software for Peggy User Guide K562 1ma ml anti ERK 1 2 Cell Signaling Exposure Multi C1 10 40 MW kDa page 210 Chapter 8 Size Assay Data Analysis Selecting Data Viewing Options The graph view menu provides multiple user options for changing what type of electrooherogram data is displayed To access the view menu click the down arrow next to the graph pane toolbar L Graph gt PA Image FJ Lane FB i o i Samples ee Sample Baseline Corrected 1300 Baseline Fit 1200 Baseline Points 1100 Fit 1000 Fit Baseline Corrected 7 300 Standards o 800 2 700 5 600 5 500 O 400 300 200 100 0 100 12 40 66 90 116 180 MW kDa A check mark next to the menu option indicates it is currently selected Multiple options can be selected at a time NOTE Unless otherwise noted graph view menu options are available for sample data only Compass Software for Peggy User Guide Changing the Electropherogram View page 211 e Sample Clicking this option will display raw uncorrected sample data Sample Baseline Corrected Clicking this option will display sample data with the baseline sub tracted zeroed This is the default view o Cc o oo E E E E a 000 200 200 700 600 500 400 300 200 z Compass Software for Peggy User Guide page 212 Chapter 8 Size Assay Data Anal
284. sed as a starting point for new assays will display Click Peggy Charge The Peggy template assay and default settings will display in the Assay screen Compass Software for Peggy User Guide page 68 Chapter 4 Charge Assays File Edit Instrument Window Help Run Charge Fii Add v Remove Ey lt XOs8s12345170 Cyclel Sample Separation Immobilization Time sec 100 0 Primary Ab Time min 120 0 Secondary Ab Time min 60 0 Tertiary Ab Time min 10 0 Detection VOSErAc TOMmmMOOOy VOZErxAcC TOAMMOOOy Step 2 Assign Assay Plate Reagents Optional 1 Click on the Layout tab This pane displays the default row locations of where each reagent should be placed on the assay plate NOTE Up to 96 different samples or conditions lysates proteins antibodies or incubation times can be assayed using the full eight cycles to accommodate experimental needs Compass Software for Peggy User Guide Creating a New Assay page 69 RowA Samples VOZEr Ac 0p nm g 0 T p Ozn AZt rono 0m e RowB Primary Antibody Row C Secondary Antibody e RowD Tertiary Antibody optional gt RowE Luminol S Peroxide mix NOTE For details on sample reagent and assay plate preparation please refer to the product insert pro vided with the Simple Western kits 2 If needed well assignments can be modified as described below Any row assignments changed in the Layout pane are updated in the Protocol pane automatically
285. selected the following pop up box appears to let you enter a specific capillary number or range of capillaries woven Enter cycle and capillary descriptor Examples 2 3 1 1 12 Cycle 2 capillary 3 and cycle 1 capillaries 1 through 12 1 3 6 8 10 Cycles 1 through 3 capillaries 6 amp and 10 Cancel 6 Ifyou need to change the peak names group used for a data set click the cell in the Settings column and click the down arrow Select a group from the drop down list Compass Software for Peggy User Guide Chapter 9 Charge Assay Data Analysis page 376 Apply Settings Settings ERKI ERKI 7 Repeat the previous steps to apply other groups to specific run data 8 To remove a data set click on its cell in the Apply To column then click Remove Compass Software for Peggy User Guide Peak Names Settings page 377 9 Click OK to save changes Named peaks will be identified with a peak name label in the electrophero gram and virtual blot and will be color coded in the peaks and capillaries tables Training data_Hi Low ERK Compass File Edit View Instrument Window Help a E Jeli fg Sample Primary Cyc Hi ph ERKI 2 1 Low p ERK1 2 pErk1 Erk1 pERK2 v Hi ph ERK1 2 ppERK2 Low p ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Low p ERK1 2 Hi ph ERK1 2 Low p ERK1 2 Hi ph
286. sers pfung Documents Compass Runs lt Back Next gt Cancel i Click Start to begin the run Instrument status will change to running and the stop button and progress bar will display Compass Software for Peggy User Guide Starting a Run page 59 2012 05 30_ 15 52 06 DEX70K_2mil Running Stop LL Wed 3 58 PM Thu 11 28 AM a The run will continue until complete 14 19 hours depending on the assay Step 3 Post Run Procedures 1 Empty the capillary discard tray 2 Remove the assay plate 3 Dispose of the assay plate and capillaries Disposal will depend on the samples that have been assayed If sample origins are unknown ProteinSimple recommends that used capillaries and plates be disposed of in biohazard waste WARNING SHARPS HAZARD The capillaries may present a potential sharps hazard Dispose of used capillaries in biomedical waste sharps containers IWARNING BIOHAZARD A Samples and waste bottle contents should be handled by procedures recommended in the CDC NIH manual Biosafety in Microbiological and Biomedical Laboratories BMBL The manual is available from the U S Government Printing Office or online at http www cdc gov biosafety publications bmbl5 Depending on the samples used waste bottle contents may constitute a biohazard Use precaution when emptying the waste bottle Dispose of waste bottle contents in accor dance with good laboratory practices and loca
287. settings group selected to specific run data as follows All When this option is selected peak names group settings will be applied to all capillaries in the run file Blocking reagent name All blocking reagent names entered in the assay template Assay screen will display in the drop down list otherwise the default name of Blocking will be shown Select aname to apply group settings to all capillaries that use this reagent name in the run file Primary antibody names All primary antibody names entered in the assay template Assay screen will display in the drop down list otherwise the default name of Primary will be shown Select a name to apply group settings to all capillaries that use the primary antibody name in the run file Attributes All primary antibody attributes entered in the assay template Assay screen will dis play in the drop down list Select an attribute to apply group settings to all capillaries that use this attribute in the run file Cycle 1 8 When this option is selected group settings will be applied to all capillaries in the cycle Custom settings Lets you choose specific capillaries to apply the group settings to When this option is selected the following pop up box appears to let you enter a specific capillary number or range of capillaries E Custom Settings Enter cycle and capillary descriptor Examples 2 3 1 1 12 Cycle 2 capillary 3 and cycle 1 capillaries 1 through 12 1 3 6 6 10
288. steps will also help you identify and correct any issues Step 1 Review the Fluorescent Sizing Standards Movie All capillaries should have three fluorescent sizing standards To verify the standards separated in the capil lary correctly 1 When the run has completed click the Run Summary screen tab 2 Click the Separation tab and play the movie this will be the fluorescent standards separation Compass Software for Peggy User Guide page 296 Chapter 9 Charge Assay Data Analysis amp 2012 03 05_11 51 19 HelaControlERKassay Compass Co let File Edit Instrument Window Help Assay Run Summary i Analysis Run 2012 03 05_11 51 19_HelaControlERKassay E Separation lt IV Plot PY Status ii Run 2012 03 05_11 51 19_HelaControlERKassay Path C Users pfung Documents ProteinSimple 2012 03 05 11 51 19 HelaControlERKassay cl Assay HelaControlERKassay Schedule Overlapping with hold Instrument PLOOO4 PLOO04 Stared Mon 11 56 AM Mar 5 2012 PST Completed Tue 6 35 AM Mar 6 2012 PST Cycle 3 Cycle Sample Sep Hold 1 2 a Detect Results TY Tan rM zT Cal r t 1 LH Simri 11 56 AM 12 01PM 1 10PM 6 10 PM 6 36 PM 8 45 PM 9 53PM 10 27PM TY wa 2 TY a T m po 2 Lil e Ta ieai D o Simier 1 10 PM 1 14 PM 2 24 PM 7 27 PM 7 53PM 10 02PM 11 10PM 11 44 PM TY Tar r zT m 3 Lii _ aad eat ahd Uem 2 24 PM 2 28 PM 3 37 PM 8 46 PM 9 13PM 11 22PM 12 30AM 1 04 AM
289. sults for each immunodetected protein are shown in the peaks and capillaries tables NOTE The reported pl for immunodetected sample proteins in Compass may vary slightly trom predicted pls based on sample buffer and assay conditions Compass Software for Peggy User Guide page 280 Chapter 9 Charge Assay Data Analysis For information on checking and identifying sample peaks see Step 4 Checking Samples on page 301 To view standards data Click Show Standards in the View bar or select View in the main menu and click Standards DemoData_Charge_HeLa_ERK12 Compass File Edit View Instrument Window Help Assay C3 Run Summary wer 2m v Ie a Sample y Standards High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 High phospho HeLa in 5 8 Low phospho HeLa in 5 8 Fluorescence S B 8 amp 8 g Std 6 0 Std 6 4 Std 7 0 y y y Position Primary Cap Peak ERK1 2 c14 ERK1 2 C14 ERK1 2 Cc1 4 ERK1 2 Ci 4 ERK1 2 c14 ERK1 2 c14 c14 FRK1 Ci 4 Compass Software for Peggy User Guide Viewing Run Data page 281 Data in this view is for the fluorescent standards only These standards are premixed with each sample prior to analysis Graph view data displays electro
290. ta auth user Deny analysis editing llow instrument administrati auth user Deny copy export of data llow instrument control auth user Deny instrument administrati llow plate editing auth user Deny instrument control llow protocol editing auth user Deny plate editing llow sign off of data auth user Deny protocol editing i auth user Deny sign off of data Choose all Remove all Delete Save and add another Save and continue editing Save Adding Admin Users To create a user with administrator permissions 1 Follow the steps described in Adding Non admin Users on page 412 to create the admin user 2 Under permissions select Staff status and Superuser status Active Designates whether this user should be treated as active Unselect this instead of deleting accounts Staff status Designates whether the user can log into this admin site Superuser status Designates that this user has all permissions without explicitly assigning them 3 Assign the admin user to a group Compass Software for Peggy User Guide page 418 Chapter 11 Compass Access Control and 21 CFR Part 11 Compliance NOTE Selecting Superuser status enables server permissions only Admin users must be also be assigned to a group to in order to have Compass permissions Resetting User Passwords To reset a user password 1 Select Users from the Site Administration home page then select the us
291. ta Analysis NOTE Peggy can run up to eight cycles 12 capillaries per cycle in an experiment Each cycle is designated in the Experiment summary Cap Capillary number S Well on the assay plate used for sample 1 Well on the assay plate used for blocking buffer Antibody Diluent 2 Well on the assay plate used for primary antibody 3 Well on the assay plate used for secondary HRP conjugate Graph Pane Electropherogram Data Click the Graph tab to view data for immunodetected sample proteins fluorescent standards or capillary registrations Data for samples is shown in the following example and immunodetected proteins are dis played as peaks kx Graph gt H Image Lane F BE my Samples 1300 ERK2 12004 11004 1000 K562 C1 10 900 3 70 5 eo 5 500 gt 400 ERKI a 300 200 100 07 100 4 40 66 90 116 180 MW kDa More Graph view options will be described in more detail in Changing the Electropherogram View on page 196 Compass Software for Peggy User Guide How Run Data is Displayed in the Analysis Screen page 141 Image Pane Capillary Separation Image Data Click the Image tab to view final separation images of immunodetected sample proteins fluorescent stan dards or capillary registrations for up to 96 capillaries per experimental run Image data for samples is shown in the following example hire Graph w Image Lane Cycle 1 Sa
292. tandards or capillary registrations e Lane Displays data for immunodetected sample proteins fluorescent standards or capillary registra tions as bands in individual lanes This virtual blot like image is similar to traditional blot results e Peaks Lists the tabulated results for immunodetected sample proteins and information for identi fied fluorescent standards and capillary registrations Capillaries Displays a list of the immunodetected sample proteins Compass named automatically using the user defined peak name analysis parameters Compass Software for Peggy User Guide Analysis Screen Overview page 133 2012 03 05_11 51 19 HelaControlERKassay Compass o ss File Edit View Instrument Window Help eesGia e E Assay CA Run Summary EE Experiment N S ma E Graph E Image FJ Lane a la zZ i Sample Primary Gi es Ka o SEPELE a ae ae TPP LPP L ELE LEP ES biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki Block hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki biotin Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela Blocki hela
293. ted click in the first cell in the MW column in the ladder table Ladder Capillary Position 200 500 600 700 200 900 Pes Enter the molecular weight in kDa for the ladder standard Compass Software for Peggy User Guide page 256 Chapter 8 Size Assay Data Analysis Ladder Capillary 200 500 700 200 900 Fi 13 Click in the first cell in the Position column Ladder Capillary Mw Fit 15 40 66 gi 116 180 lt A A a A s Enter the position of the ladder standard peak e es ee MW Position Fit 15 205 40 500 66 90 700 116 800 180 900 lt lt I A a EI J NOTE adder peak positions are relative to each other Only the difference in their position is used to help identify the ladder peaks When entering ladder peak information for the first time review the ladder data in the Analysis screen to find the correct peak position Compass Software for Peggy User Guide Standards Settings page 257 14 Repeat the steps above for the remaining ladder standards in the table To add another ladder standard Click Add under the table then modify the information in the new row Toremove a ladder standard Select its row and click Remove 15 Select which ladder standards should be used for molecular weight determination of sample proteins by clicking the checkbox in the Fit column All ladder standards are typically used for fit Ladder Capillary MW
294. ted as secondary antibody in the Layout tab can be selected in the Well Row drop down menu 7 Additional exposures can be collected in the assay if desired To do this click the white arrow next to Detection to expand the row Click the cell in the exposure column next to Detection Profile to open the Detection Profile window Additional times can be added to the protocol by clicking the Add button entering the values and selecting OK Compass Software for Peggy User Guide page 34 dr gt Protocol Notes Cycle 1 Cycle 2 Separation Matrix Stacking Matrix Sample Separation Time min 40 0 40 0 Separation Voltage volts 250 250 Matrix Removal Blocking Time min 25 0 25 0 Primary Ab Time min 120 0 120 0 Secondary Ab Time min 60 0 60 0 Detection Well Row Fl F1 Detection Profile 7 Exposure 6 Exposures 1 Detection Profile Cycle 3 40 0 250 25 0 120 0 60 0 Fl 6 Exposures 8 Modify any other protocol parameters as needed NOTES Cycle 4 40 0 120 0 Cycle 5 Cycle 6 6 Exposures Chapter 2 Size Assays Add Remove Cycle 7 Cycle amp 40 0 40 0 250 250 25 0 25 0 120 0 120 0 60 0 60 0 Fl Fl 6 Exposures 6 Exposures Edit For more information on changing protocol step parameters other than incubation times contact ProteinSimple Technical Support When a protocol is being edited an asterisk will appear in the Protocol tab to indicate changes have been ma
295. teinSimple recommends using the default parameters for Simple Western assays However users can modify any parameters as needed then click OK For detailed information on analysis parameters please refer to Compass Analysis Settings Overview on page 219 Compass Software for Peggy User Guide Making Changes to an Existing Assay Making Changes to an Existing Assay 1 Select File in the main menu and click Open Assay Edit Instrument Window Help a Open Assay p Protein Sizing Save Protein Test Save As Peggy Size Simple Western Demo Plate Import Protecal Simple Western Import Template Export Protocol Export Template Print p n mo Dp Exit h Separat gt Separat gt Matrix f gt Blockin gt Primary page 43 2 Alist of the last assays opened will display Select one of these assays or click Browse to open the assay folder and select a different assay 3 Follow the steps in Creating a New Assay on page 25 to make changes and save the assay Switching Between Open Assays If more than one run file is open you can switch between viewing the assays for each run To do this 1 Click the down arrow in the run box File Edit Instrument Window Help Ready New Run pl Run HeLa IKb test ki HUT78 Screen Lay HeLa IKb test 2 Select the run for the assay you want to view from the drop down list Compass Software for Peggy User Guide
296. tes that a capillary registration was not identified prop erly This can be resolved by manually identifying the registration peak Please refer to Step 3 Checking Capillary Registrations on page 299 for details Rolling the mouse over the icon displays warning details Compass Software for Peggy User Guide Checking Your Results page 295 e Manual correction of registrations notification Indicates the user modified the capillary registration data manually Rolling the mouse over the icon displays the type of data modification made Peak fit warning Indicates that a peak cannot be fit properly This is typically caused either by a very small peak in the electrooherogram or a peak that is very close to the end of the pl range This can be resolved by removing the peak Please refer to Step 4 Checking Samples on page 301 or Step 4 Checking Samples on page 301 for details Rolling the mouse over the icon displays warning details 4s Kitlow pho anti H 2 4 Kitlow pho anti B 2 5 Peak Fit Warning Too many iterations Checking Your Results Compass detects immunodetected proteins fluorescent standards and capillary registrations and reports results automatically with no user interaction However ProteinSimple recommends users review their data as outlined in this section as good general practice to ensure accurate results If a data warning is presented in the experiment pane the following
297. the View bar Verification that sample proteins have been correctly identified can be done in either the graph or lane panes but manual corrections must be done in the graph pane Graph Pane a Click Single View in the View bar b Click on the row in the experiment pane that contains the sample you wish to check then click the Graph tab Compass Software for Peggy User Guide Checking Your Results page 173 PE u 2 fg E Assay C3 Run Summary 8 ie Graph Bl Image E Lane 7 el jm Primary in Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki e Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki Blocki in Blocki Blocki Blocki Blocki Blocki Blocki Rlacbli Chemiluminescence 288828 822858 amp 8 BE 40 MW kDa _EE Capillaries Sample ulin Cap Peak Name Position MW kDa Height Area Area Width hela Blocki 2 F amp F F amp F FF Fe WhWwW Www sgnwwnsnwnnnnnn nr nN NNN NN FR RRR RP RP RP RF ee If sample peaks are not identified correctly they can be manually corrected as follows If an incorrect peak is identified as a sample peak Right click the peak
298. ther users that the instru ment is available being serviced or when an error has been cleared To do this 1 Select Edit in the main menu and click Preferences and click Twitter in the preferences list 2 Click Tweet Message 3 Enter atest message and click OK The tweet will be received by any users following the Twitter account Peggy uses Compass Software for Peggy User Guide page 400 Chapter 10 Setting Custom User Preferences Compass Software for Peggy User Guide page 401 Compass Access Control and 21 CFR Part 11 Compliance Chapter Overview Overview Enabling Access Control Logging In to Compass Saving Changes Signing Files Instrument Command Log Run File History Troubleshooting Problems and Suggested Solutions Authorization Server Compass Software for Peggy User Guide page 402 Chapter 11 Compass Access Control and 21 CFR Part 11 Compliance Overview The Compass Access Control feature can be used to help satisfy the 21CFR Part 11 data security require ments when using Simple Western instruments When Access Control is enabled and the Authorization Server has been installed see Authorization Server on page 410 Users are required to log in to Compass when the software is launched A history of all actions is maintained Data files are signed and encrypted to prevent unauthorized changes e g all files are controlled Each instrument maintains a history of user commands Each assay and data
299. tings for sample proteins Compass can automatically label sample peaks in the run data associated with specific blocking reagent names primary antibody names or attributes To access these settings select Edit in the main menu and click Analysis then click Peak Names in the options list NOTE Settings can be modified in an assay prior to starting a run or ina run file once the run has finished executing Analysis changes made to an executing run will not be saved to the final run file Analysis Simple Western ERK Demo Sk type filter text Peak Names Advanced Images Analysis Settings Analysis Settings Peak Names 1 Peak Names 1 Peak Names Mi Color Range Show Standards 3 10 Apply Settings Settings Remove Restore Original Click Import to import an analysis settings file This will be explained in more detail in Importing Analysis Settings on page 261 Click Export to export the current analysis settings file This will be explained in more detail in Exporting Analysis Settings on page 261 Click Restore Original to restore Compass default settings Compass Software for Peggy User Guide Peak Names Settings page 239 Click OK to save changes and exit Click Cancel to exit without saving changes Peak Names Analysis Settings Groups Peak name settings are saved as a group and multiple settings groups can be created Specific group set tings can then be applied to individual cap
300. trix Stacking Matrix Sample Separation Time min 40 0 40 0 40 0 40 0 40 0 40 0 40 0 40 0 Separation Voltage volts 250 250 250 250 250 250 250 250 Matrix Removal Blocking Time min 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 Primary Ab Time min 120 0 120 0 120 0 120 0 120 0 120 0 120 0 120 0 Secondary Ab Time min 60 0 60 0 60 0 60 0 60 0 60 0 60 0 60 0 Detection VOZZrZze I0nmoO0o f VOZErAC TOAMMOOGy Compass Software for Peggy User Guide Assay Screen Overview page 21 Software Menus Active in the Assay Screen The following software menus are available File Edit Instrument when Compass is connected to Peggy Window Help The File and Edit menu options specific to the Assay screen are described next File Menu The following File menu options are active Edit Instrument Winda New Assay d Open Assay k Save Save As Import Protecal Import Template Export Protocol Export Template Print p Exit New Assay Creates a new assay from a starter template Open Assay Opens an existing assay Save Saves the open assay Save As Saves the open assay under a different file name Import Protocol Imports a saved protocol file into an assay Import Template Imports a saved template file into an assay Export Protocol Exports the current protocol file for future use Export Template Exports the current template file for future use Print Prints the informat
301. troughs have already been cleaned click OK to continue Peggy s status will change to cleaning and the stop button and the cleaning progress bar display The Assay screen provides cleaning status details Cleaning T Run Summary ik Analysis la Cleaning O O p Sat 4 45 PM Sat 4 53 PM When cleaning is complete instrument status will change to Ready File Edit Instrument Window Help Cleaning After a Run Error Additional cleaning steps are required if an error occurs that stops the run When this happens the red Error status light on Peggy s front panel will come on Click on the Reset button displayed in Compass software The following instructions will appear Compass Software for Peggy User Guide Instrument Control page 123 amp Cleanup Make sure the separation tray troughs are clean before clicking OR To clean the troughs click Cancel and use the Open Trays menu to access the separation tray troughs Cleaning the troughs may require soaking them with distilled water If the troughs have already been cleaned click OK to continue If the troughs in the separation tray are empty click on OK and proceed with Cleanup on page 121 If Running Buffer is present in the separation tray click on Cancel and manually remove the buffer Evapora tion of the Running Buffer will result in a highly viscous residue which the automatic cleaning feature cannot remove To remove the Running Buffer
302. trument Window Help Open Run Simple Western ERK Demo Add Run 2011 08 31_16 38 23_test nee 3 ab run Close All ee jE 2011 09 01_16 41 02 i Browse 2 Alist of the last 10 runs opened will display Select one of these runs or click Browse to open the Runs folder and select a different file Opening Multiple Run Files 1 To open the first run file select File in the main menu and click Open Run Edit Instrument Window Help Open Run Simple Western ERK Demo Add Run 2011 08 31_16 38 23 test Close 3 ab run Close All DemoData 2011 09 01_16 41 02 Exit Path CAU geep Browse Assay simple Western Demo Assay 2 Alist of the last 10 runs opened will display Select one of these runs or click Browse to open the Runs folder and select a different file 3 To open another run file select File in the main menu and click Add Run Compass Software for Peggy User Guide page 110 Chapter 6 Run Status File Edit Instrument Open Run Add Run 2012 03 05_11 51 19_HelaControlER Kassay Close Close All Exit 4 Alist of the last 10 runs opened will display Select one of these runs or click Browse to open the Runs folder and select a different file 5 Repeat the last two steps to open additional runs Compass Software for Peggy User Guide Viewing File and Run Status Information page 111 Viewing File and Run Status Information Information specific to each run f
303. ture entry in the file History User Name Message Comment 04 15 2013 9 48 AM b RunStart2013 04 15 09 49 23 Simon_short 05 10 2013 4 22 PM steveg Saved analysis changes Changed Baseline fr 05 10 2013 4 22 PM User Saved analysis changes Changed Baseline from 1 0 to 2 0 Signing Files Select e Signature from the File menu to add an electronic signature to a file Add your signature to this file Comment Approved Compass Software for Peggy User Guide page 407 Instrument Command Log The signed entry will be added to the file History with the meaning of the signature entered in the com ment such as Approved or Verified Comment User Name Message RunStart2013 04 15 09 49 28 Simon_short 04 15 2013 9 48 AM b 05 10 2013 4 22 PM steveg Saved analysis changes 05 10 2013 4 44 PM steveg e Signature applied Changed Baseline fr Approved Time 05 10 2013 4 44 PM Message e Signature applied Comment Approved Instrument Command Log The Instrument Command Log can be viewed at any time by selecting the Instrument menu and clicking Properties and then clicking the Command Log button Name Peggy PLOOOS Location Network Name pl0005 local Type Peggy Network Address 10 1 2 173 Serial Number PLOOO5 Instrument Software 2 1 18354 Instrument Date and Time 2013 05 10 17 03 02 07 00 et Compass Software for Peggy User Guide page 408 The Command Log lists all the commands
304. u and click Open Assay rl Ready New Run P File Edit Instrument Window Help New Assay Open Assay MEK assay Protein Test Save Save As Peggy Charge Peggy Charge 1017 Import Proteceal Erk Assay Import Template Export Protocol Export Template Print r TnM OD p Exit b A list of the last five assays opened will display Select one of these assays or click Browse to open the Assay folder and select a different assay c Alternatively choose New Assay and select Peggy Charge to get the default Peggy assay condi tions d The Start button will display START BUTTON this indicates an assay is already loaded a Go to the Assay screen and verify this is the assay you want to use If not select File in the main menu click Open Assay and select another assay 2 Click Start This will launch the Start Run Wizard NOTE If the manifold was not cleaned prior to starting the run a message indicating this will display If this occurs click Yes to cancel the run and perform the manifold cleaning Compass Software for Peggy User Guide page 98 Chapter 5 Running a Charge Assay on Peggy 3 Page 1 Check Water and Waste The fluid levels in the accessory module bottles will be checked by the software If the levels in both bottles will allow Peggy to complete the run the wizard screen will dis play Water Level OK and Waste Level OK messages Click Next to proceed Water le
305. ultiple wells or a row To select individual wells press and hold the Control key and then select individual wells To select a sequential set of wells or a full row press and Compass Software for Peggy User Guide page 82 Chapter 4 Charge Assays hold the Shift key then select the first well and last well Next right click and select Edit or click Edit in the upper right corner of the pane The following box will display Well Content Name fo Notes Attribute Well Content Name HeLa Notes Lysate Attribute 1 mg mL a Click OK The new information will display in the selected wells Compass Software for Peggy User Guide Creating a New Assay page 83 NOTE When annotations are being edited an asterisk will appear in the Template tab to indicate changes have been made and should be saved Only Name and Attribute will be used by Compass to annotate the data Compass Software for Peggy User Guide page 84 Chapter 4 Charge Assays Step 7 Save the Assay 1 Select File from the main menu and click Save As Enter the assay name and click Save gt w Organize v New folder Kr Favorites Documents library BB Desktop Assays m Downloads Name Date modified E Recent Places Arrange by Folder Type SW long incubation assay 9 29 2011 3 27 PM Compass Assay File Simple Western 2 assay 9 29 2011 3 24 PM Compass Assay File Test assay assay 9 29 2011 3 21 PM
306. up shown contains the Compass default analysis settings Users can make changes to this group and create new groups To view settings for a group click on the group name in the analysis settings box Creating a New Peak Fit Group 1 Select Edit in the main menu and click Analysis then click Peak Fit in the options list 2 Click Add under the analysis settings box A new group will be created Analysis Settings Peak Fit Peak Fit 2 3 Click on the new group and enter a new name Analysis Settings Peak Fit STAT peak fit 4 Modify range baseline or peak find parameters as needed 5 To use the new group as the default peak fit settings for the run file data click the arrow in the drop down list next to Default then click the new group from the list Peak fit settings in the new group will then be applied to the run data Compass Software for Peggy User Guide Peak Fit Analysis Settings page 363 Analysis Settings Peak Fit STAT peak fit Default Peak Fit Override STAT peak fit 6 Click OK to save changes Changing the Default Peak Fit Group 1 Select Edit in the main menu and click Analysis then click Peak Fit in the options list 2 Click the arrow in the drop down list next to Default then click a new default group from the list Analysis Settings Peak Fit STAT peak fit Peak Fit Override STAT peak fit 3 Click OK to save changes Peak fit settings in the group selected will be ap
307. ur Results Compass detects immunodetected proteins fluorescent standards and capillary registrations and reports results automatically with no user interaction However ProteinSimple recommends users review their data as outlined in this section as good general practice to ensure accurate results If a data warning is presented in the experiment pane the following steps will also help you identify and correct any issues Step 1 Review the Fluorescent Sizing Standards Movie All capillaries should have three fluorescent sizing standards To verify the standards separated in the capil lary correctly 1 When the run has completed click the Run Summary screen tab 2 Click the Separation tab and play the movie this will be the fluorescent standards separation Compass Software for Peggy User Guide page 164 Chapter 8 Size Assay Data Analysis amp 2012 03 05_11 51 19_HelaControlERKassay Compass Co fE File Edit Instrument Window Help E Assay TE Run Summary k Analysis Run 2012 03 05_11 51 19_HelaControlERKassay Separation b lt IV Plot Y Status m Run 2012 03 05_11 51 19_HelaControlERKassay Path Ci Users pfung Documents ProteinSimple 2012 03 05 11 51 19 HelaControlERKassay cl Assay HelaControlERKassay Schedule Overlapping with hold Instrument Sally Sally PLOOO4 PLOOO4 Started Mon 11 56 AM Mar 5 2012 PST Completed Tue 6 35 AM Mar 6 2012 PST Cycle 3 Cycle Sample Sep Hold 1 2 3 Detect Results
308. ur default assay for STATS analysis for samples run in triplicate NOTE When notes are being edited an asterisk will appear in the Notes tab to indicate changes have been made and should be saved Step 5 Select a Schedule Optional Peggy can execute cycles serially or in parallel To choose an option select Edit and click Schedule eS Schedule Serial O Overlapping Overlapping with hold a Gee Serial Each cycle is executed in sequential order and each cycle completes before the next one starts Selecting this option result in the longest run times e Overlapping Cycle steps are executed in parallel if timing permits shared use of the separation tray and robot Each cycle will run through to completion once started This option offers a reduction in overall run time without affecting the individual run time for each cycle e Overlapping with Hold default Each cycle executes through the separate step in sequence and then enters a variable length hold step where capillaries remain in the incubation tray More sample stability is provided with this option as proteins are locked to the capillary wall and therefore more stable than samples on the assay plate This option also results in the most signif icant reduction in overall run time Compass Software for Peggy User Guide Creating a New Assay page 79 NOTE ProteinSimple recommends using the overlapping with hold option For more information on when t
309. ve and continue editing Save After adding a new user more information can be added Compass Software for Peggy User Guide page 414 Chapter 11 Compass Access Control and 21 CFR Part 11 Compliance Change user ProteinSim e gt C 4 10 1 2 18 8000 admin auth user 3 Home gt Auth gt Users gt steveg Change user Username steveg Required 30 characters or fewer Letters digits and _ only Password algorithm pbkdf2_sha256 iterations 10000 salt Rik6oj hash ane ee Raw passwords are not stored so there is no way to see this user s password but you can change the password using this form First name Last name Email address Permissions All users can log in to Compass but the commands available within Compass are controlled by Permission settings Commands a user does not have permission to use will be disabled After user permissions have been changed on the server the user must close and re open Compass to use the new permissions Users can belong to groups that have multiple permissions such as Manager or Scientist Compass Software for Peggy User Guide Authorization Server Change user ProteinSim X gt Q O 10 1 2 18 38000 admin auth user 10 Permissions Active Designates whether this user should be treated as active Unselect this instead of deleting accounts _ Staff status Designates whether the user can log into this admin site
310. vel OK Waste level OK r Start Run EE Check water and waste Make sure there is enough water and room in the waste bottle The run will be terminated if the water bottle empties or the waste bottle fills during the run NOTE If the waste level is too high or the water level is too low to complete the run messages to indicate one or both will be presented in this screen If this occurs fill or empty each bottle as indicated using the procedures outlined in the Peggy User Guide When this is complete the error status will be automatically updated and allow the Start Run Wizard to proceed Compass Software for Peggy User Guide Starting a Run page 99 4 Page 2 Replace Sponge A new sponge should be used each time a new experimental run is started Discard the old sponge and put a new sponge in the manifold wash station a Start Run X Replace Sponge Put a new sponge in the manifold wash station 5 Page 3 Start The resource tray will automatically open Fill the Wash Buffer Anolyte and Catholyte cups and insert the capillary box in the primary capillary box position Add a secondary capillary box if more capillaries are required Click Next when finished The resource tray will close NOTE Make sure enough capillaries are loaded to complete the run A capillary box should be inserted in the primary position first and if additional capillaries are needed use the secondary position
311. will no longer identify it as a sample peak in the electrooherogram and the peak data will be removed in the results tables e To setan unidentified peak as a sample peak Right click the peak in the electropherogram or peaks table and select Add Peak Compass will calculate and display the results for the peak in the results tables and identify the peak in the electrooherogram NOTE To remove sample peak assignments that were made manually and go back to the original view of the data right click in the electropherogram and click Clear All c Repeat the previous steps for the remaining sample rows in the experiment pane to make sure all sample proteins are identified correctly Compass Software for Peggy User Guide Checking Your Results page 303 Lane Pane a Click either Single View or Multiple View in the View bar b Click on the row in the experiment pane that contains the sample you wish to check then click the Lane tab To view sample protein band labels roll the cursor over the individual bands If sample bands are not identified correctly they must be corrected in the graph pane as described in the pre vious section DemoData_Charge_HeLa_ERK12 Compass _o E File Edit View Instrument Window Help i B E E fg v E Assay cy Run Summary E Experiment u E Graph BA Image F e OK BE Sample Primary Cycle amp S Ra 2 2 2 2 a amp Ki Pa FP PP FPL PLS GECEEEELLEE High phos E
312. xtension Compass Software for Peggy User Guide page 19 Chapter 2 Size Assays Chapter Overview Assay Screen Overview Reagent Color Coding Opening an Assay Creating a New Assay Making Changes to an Existing Assay Switching Between Open Assays Creating a Template Assay Viewing and Changing the Detection Exposures Copying Protocols and Templates Printing Protocols and Templates Importing and Exporting Protocols and Templates Compass Software for Peggy User Guide page 20 Chapter 2 Size Assays Assay Screen Overview The Assay screen is used to create view and edit assays To access this screen click Assay in the screen tab EA Assay chy Run Summary g Analysis Assay Screen Panes The Assay screen has four panes e Layout Displays a map of the assay plate and shows where assay reagents will be located e Protocol Lists individual assay protocol steps and parameters that Peggy will execute for each of the 12 capillaries simultaneously Notes Lets you enter specific assay information that is saved with the assay and can be used for future reference Template Enter annotations for the individual well and row reagents in the assay plate Size Compass File Edit Instrument Window Help Pg Run Summary fe Analysis m Run Size HE Fue x lt XOs12345L1TH5 Add v Remove Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle 8 Separation Ma
313. yout Software Help Select Help and click Help Contents to view the Compass Help file Checking for and Installing New Versions of Compass Compass can automatically check to see if a newer version of software is available To do this 1 Make sure the computer being used has an active internet connection 2 Select Help and click Check for Updates If an update is found the following screen will display Update Available Updates aA E 2S a Si A new update has been found for Compass Compass 2 3 1 3 Click Finish to start the download and install the update 4 Follow the on screen instructions to complete the software installation 5 Reboot the computer before using the new version of Compass Compass Software for Peggy User Guide Viewing Release Notes page 15 Viewing Release Notes Select Help and click Release Notes to view feature updates and bug fixes for new and past versions of Compass ProteinSimple recommends users review these notes whenever a software update is installed Release Notes txt Notepad 2 8 File Edit Format View Help Compass 2 4 Release Date November 15 2012 1 Support for running new instrument Peggy Compass 2 2 1 Release Date February 28 2012 1 Support for running new instrument Sally support multi cycle sizing assay Compass 2 1 Release Date November 30 2011 1 Lane view improvement for sizing data Compass 2 0 Release Date October 31 201
314. ype filter text Images Advanced Images Cycle Luminescence Peak Fit Peak Names s All Multi Image Analysis KA 1 Multi Image Analysis z 2 Multi Image Analysis z 3 Multi Image Analysis 4 Multi Image Analysis z 5 Multi Image Analysis v 6 Multi Image Analysis 7 Multi Image Analysis x 8 Multi Image Analysis z Import Export Restore Original Cane Click Import to import an analysis settings file This will be explained in more detail in Importing Analysis Settings on page 261 e Click Export to export the current analysis settings file This will be explained in more detail in Exporting Analysis Settings on page 261 Click Restore Original to restore Compass default settings Click OK to save changes and exit Click Cancel to exit without saving changes Compass Software for Peggy User Guide Images Analysis Settings page 229 Exposure Settings The exposure used for the sample data displayed in the Analysis screen is shown in the All box Cycle Luminescence All Multi Image Analysis 1 Multi Image Analysis 2 Multi Image Analysis 3 Multi Image Analysis 4 Multi Image Analysis 5 Multi Image Analysis 6 Multi Image Analysis NOTE Peggy runs up to eight cycles 12 capillaries per cycle The individual exposure setting selected for a specific cycle is applied for all 12 capillaries in that cycle unless selected for All cycles e Multi lmage Analysis Sample
315. ysis Baseline Fit Clicking this option will display the calculated baseline for the raw sample data In the example that follows both Baseline fit and Sample are selected NOTE This option is selected automatically when Baseline Fit is selected in graph options K562 C1 10 D TI T TI a E 3 E 40 MW kDa Baseline Points Clicking this option will display regions of the electropherogram considered to be at baseline In the example that follows both Baseline Points and Sample are selected Compass Software for Peggy User Guide Changing the Electropherogram View page 213 NOTE This option is selected automatically when Baseline Fit is selected in graph options PSEBEES o ce o we E a E f 50116 Compass Software for Peggy User Guide page 214 Chapter 8 Size Assay Data Analysis e Fit Clicking this option will display the bounding envelope of the fitted peaks as calculated by the software for the raw sample data In the example that follows both Fit and Sample are selected kn Grap ha E Image Era Gale B 7 ml Samples Biotinylated Ladder C1 1 D La T o we a E 3 E a D MW kDa Compass Software for Peggy User Guide Changing the Electropherogram View page 215 Fit Baseline Corrected Clicking this option will display the fitted peaks as calculated by the soft ware for the sample baseline corrected data In the example that follows both
316. ysis settings to be imported into other assays or run files at a later time rather than having to re enter settings manually Importing Analysis Settings NOTE Importing an analysis settings file populates the settings in all Analysis pages 1 Open the run file or assay you want to import analysis settings to Compass Software for Peggy User Guide Importing and Exporting Analysis Settings page 387 2 Select Edit in the main menu and click Default Analysis Assay screen or Analysis Analysis screen 3 Click Import on any page 4 Select a settings file settings and click OK The imported settings will display in all analysis pages Exporting Analysis Settings NOTE Exporting an analysis settings file exports the settings in all Analysis pages 1 Open the run file or assay you want to export analysis settings from 2 Select Edit in the main menu and click Default Analysis Assay screen or Analysis Analysis screen 3 Click Export on any page The following window displays Organize v New folder Fr Favorites Documents library Ez Desktop Assays ib Downloads i Recent Places Arrange by Folder v Name Date modi Type _ ERK analysis settings settings 10 16 201 SETTINGS ME Desktop _ default settings 10 16 201 SETTINGS Libraries 5 Documents E My Documents d Compass do Assays J New Assays d Runs di lt 1 File name Simple Western analysis v Save as type Analysis Setti

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