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Ettan DIGE System - McGill University

1. SDS Equilibration stock solution Reagents Quantity Final concentration Tris 1 5 M pH 8 8 10 ml 75 mM Urea MW 60 06 72 07 g 6M Glycerol 87 v v MW 69 ml 30 v v 92 09 SDS MW 288 33 4g 2 w v Make up to 200 ml with distilled water This stock solution can be stored at room temperature Stable for 6 months Add DTT or lodoacetamide for equilibration solution 1 or 2 Equilibration solution 1 Reagent Quantity Final concentration SDS equilibration buffer 100 ml stock solution DTT MW 154 2 0 5g 0 5 w v Solution should be used immediately Do not store Ettan DIGE System User Manual 18 1173 17 Edition AB 59 6 Second dimension SDS PAGE 6 7 Recipes 60 Equilibration solution 2 Note lodoacetamide should not be used in combination with saturation labeling lodoacetamide allows effective alkylation of thiols while minimizing reoxidation of the competing thiol pairs in protein samples Reagent Quantity Final concentration SDS equilibration buffer 100 ml stock solution lodoacetamide MW 185 0 459 4 5 w v Solution should be used immediately Do not store 0 5 w v Agarose overlay solution Reagent Quantity Final concentration SDS electrophoresis running 100ml buffer for Ettan DALT Low melting point agarose 0 59 0 5 w v prep 1 Bromophenol blue stock 200 ul 0 002 w v solution Mix components in a 250 ml conic
2. User Name IV Present Time Date IV Image Name IV Scan Time Date IV Image Comment Prep Time Date m Results IV Object Name IV Area M Volume T Maximum Value I Percent IT Maximum Position I Background Value Minimum Value I Background Type I Centroid T Average JT Minimum Position Standard Deviation Perimeter I Sum Width Height I Median I Comment J Sum Above Background I Pixels Above Background gt Print Format C Image and Results Results Only ora 15 Generate a volume report by clicking Analysis Volume Report in the drop down menu 16 Select all the relevant RECT in the Inspector window so that they are highlighted blue Objects IV Display I Print 17 Determine the labeling efficiency by comparing the volume of the new protein samples and the control sample which are on the same gel The labeling efficiency of these should be equivalent If labeling is comparable between the control and the new protein lysates tested then samples can now be run on 2D gels See the decision tree in Fig A 3 To investigate the cause of the problem post stain the gel with Deep Purple Protein Total Stain See Section 9 5 for information 94 Ettan DIGE System User Manual 18 1173 17 Edition AB Testing cell lysates for successful labeling A E Yes Go to 2D m Yes No Deep Purple stain Re pH re label for loading and run 1D l me Incompatible R
3. C for up to three months Cell lysis solution option 2 Reagent Quantity Final concentration Urea MW 60 06 48 0g 8M Tris 1 M not pH d 3 0 ml 30 mM CHAPS MW 614 89 4g 4 w v Make up to 100 ml with distilled water Small aliquots can be stored at 20 C for up to three months 2x Sample buffer For recipes see section 5 5 2 mM Tris 2 carboxyethy phosphine hydrochloride TCEP 2 8 mg TCEP is dissolved in 5 ml distilled water Prepare fresh solution on day of use TCEP solution is unstable and is to be used immediately Ettan DIGE System User Manual 18 1173 17 Edition AB 39 4 Sample preparation and labeling 4 9 Recipes 40 Ettan DIGE System User Manual 18 1173 17 Edition AB First dimension isoelectric focusing IEF 5 5 First dimension isoelectric focusing IEF 5 1 Ettan IPGphor 3 Isoelectric Focusing System Ettan IPGphor 3 Isoelectric Focusing System and Immobiline DryStrips are recommended for first dimension electrophoresis isoelectric focusing IEF A user friendly software Ettan IPGphor 3 Control software is provided with the Ettan IPGphor 3 Isoelectric Focusing System The software comprises several features for automatic protocol selection and protocol editing for experimental monitoring For documentation purposes it is possible to save log files and experimental parameters The use of a combination of Ettan IPGphor 3 Isoelectric Focusing System and Immobiline DryStrips gene
4. Pour off the solution and drain thoroughly 8 For samples labeled with CyDye DIGE fluor saturation dyes Do NOT use equilibration solution 2 which is containing iodoaceteamide Use equilibration solution 1 which contains DTT instead 54 Ettan DIGE System User Manual 18 1173 17 Edition AB Second dimension SDS PAGE 6 6 5 Loading of focused Immobiline DryStrips 1 Place the gels in the Ettan DALT cassette rack 2 Briefly rinse the Immobiline DryStrips by submerging them in a measuring cylinder containing SDS electrophoresis running buffer for Ettan DALT 3 Holding one end of the Immobiline DryStrip with forceps carefully place the Immobiline DryStrip in between the two glass plates of the gel Using a thin plastic spacer push against the plastic backing of the Immobiline DryStrip not the gel itself and slide the strip between the two glass plates until it comes into contact with the surface of the gel Note The strip should just rest on the surface of the gel Avoid trapping air bubbles between strip and the gel and avoid piercing the second dimension gel with the strip Note The acidic end of the Immobiline DryStrip should be on the right side of the gel when the shorter of the two plates is facing the user Note The gel face of the strip must not touch the opposite glass plate 4 Melt an aliquot of agarose overlay solution in a heating block or boiling water bath for each Immobiline DryStrips Allow the agarose t
5. e EE 5 a en en nn a i a p Phosphor Mode P 1 H H H E H H H H H H Best Sensitivity Best Resolution A 111 111111 11 1 111111 Options E F E H F E E E H F H orientation a r Press Sample K 111 111111 11 1 111111 Pixel size Focal Plane thn Broo microns x 3 mm H TT Con Fiii 2 E jiii i aE Image Analysis None B be a a 2 a a ne i i EE 1 Ee DIGE File Naming Format Ay vee Ch Gy ts ee tet teh aM es Pe hey Es aire EP hey a e KE SCAN Scanning Information r Chan Filter Laser Sensitivity PMT Splitter Total Scan Time 1 Hrs 3 Min 18 Sec 1 520BP 40 Blue2 488 nm Normal 550V Lines Per Channel 4280 2 580BP 30 Green 532nm Normal 550 Image Sis per Channel 22050 MB 3 670BP30 Red 633nm Normal 550 Fig 7 2 The Typhoon Scanner Control Multiple Sample scan window 64 Ettan DIGE System User Manual 18 1173 17 Edition AB Image acquisition 7 1 Select scan area pre defined tray area within the Tray set up area Labeled 1 in Fig 7 2 use the drop down window to select the appropriate predefined tray i e the DIGE Ettan DALT In this mode the scan area is pre defined and the software is able to recognize where individual gels will be located resulting in separate file outputs for each gel Tip The Tray Editor can be used to create tray areas for gel types other than Ettan DALT In User Select mode only the scan area is pre defined The software
6. Note Do not equilibrate Immobiline DryStrips prior to storage This must be carried out immediately before the second dimension separation Ettan DIGE System User Manual 18 1173 17 Edition AB 47 5 First dimension isoelectric focusing IEF 5 5 Recipes 5 5 Recipes 2x Sample buffer rehydration solution stock option 1 Reagent Quantity Final concentration Urea MW 60 06 10 5g 7M Thiourea MW 76 12 3 8g 2M CHAPS MW 614 89 1g 2 w v Make up to 25 ml with distilled water Small aliquots e g 2 5 ml can be stored at 20 C Stable for 6 months 2x Sample buffer rehydration solution stock option 2 Reagent Quantity Final concentration Urea MW 60 06 12g 8M CHAPS MW 614 89 1g 2 w v Make up to 25 ml with distilled water Small aliquots e g 2 5 ml can be stored at 20 C Stable for 6 months Rehydration solution Reagent Quantity Final concentration 2x Sample buffer 2 5 ml rehydration solution stock 1 or2 IPG Buffer same pH interval 50 ul 2 as the Immobiline DryStrips being rehydrated DTT 7 mg 48 Ettan DIGE System User Manual 18 1173 17 Edition AB First dimension isoelectric focusing IEF 5 DeStreak Rehydration Solution Reagent Quantity Final concentration DeStreak Rehydration 3 ml Solution 71 5025 42 IPG Buffer same pH interval 60 ul 2 as the Immobiline DryStrips being rehydrated DeStreak Rehydration Solution co
7. and this will prevent quantitative analysis being achieved Ettan DIGE System User Manual 18 1173 17 Edition AB Image acquisition 7 A target maximum pixel value of 50 000 to 80 000 is usually suitable When adjusting the voltage relatively small increments of 20 to 50 volts are recommended If only one or two spots show saturation then only slight downward adjustments to the PMT voltage setting are normally required Once the voltage has been optimized for one gel in an experiment these settings can be used for all similar gels within the same experiment The maximum pixel value should be within the specified range for all gels to enable accurate quantitation of spot volumes 7 6 Cropping using ImageQuant TL Prior to image analysis the image files should be cropped to exclude nonessential information from the image files To exclude the nonessential information ImageQuant TL Tools should be used to crop the images prior to image analysis Further cropping of the individual gel areas is normally required to remove supplementary data and can be performed in ImageQuant TL Tools 1 Define an area of interest within ImageQuant TL Tools using the dashed square button or use the Tools menu ImageQuant Tools gel 12 550 540 530 100um ds Fie Edit view Tools Image Window Help S Ba Ee J Ol fe 14 Saan av a Reduce Zoom Area Magnifier My pan select Pixel Locator Pixel Distance
8. are assembled for casting the solution will evaporate off the treated plate and coat the facing glass surface This will cause the gel to stick to both plates when it sets Note The gels will stay attached to Bind Silane treated glass during electrophoresis staining procedures scanning and storage Ettan DIGE System User Manual 18 1173 17 Edition AB Preparative workflow 9 9 4 2 Attach reference markers Reference markers are used to ensure that the correct protein spots detected during image analysis are picked Reference markers must be attached to the short treated glass plate before gel pouring It is important that the markers are appropriately placed on the treated surface of the Bind Silanized plate Take care not to place the markers where they will interfere with the pattern of protein spots in the gel The markers should be placed according to the following protocol 1 Measure the length of the treated plate edge 2 Place the marker approximately half way along this edge away from the spacer but not so far as to interfere with the protein spot pattern The marker should not touch the spacer Make sure that the markers are firmly stuck to the plate by pressing down with a lint free tissue or powder free glove 3 Repeat steps 1 and 2 for the other edge of the treated backing plate 4 When finished the markers should be in positions similar to those shown below Fig 9 2 Diagram showing the preferred position of
9. assembled gels or samples held on a glass plate Ettan DIGE System User Manual 18 1173 17 Edition AB 67 7 Image acquisition 7 5 Scan parameters and scanning 8 Make sure the instrument lid is shut Select DIGE file naming format Check the DIGE File Naming Format box labeled 8 in Fig 7 2 in the Scanner Control window to ensure that unique filenames can be generated for each scan channel Tip Using the DIGE File Naming Format option results in all files having user defined filenames All scan images from a given experiment can be saved into a single user defined folder This method of file naming and folder selection results in structures that can be directly used to crop image for subsequent DeCyder 2D analysis 9 Start the scan by clicking the Scan button labeled 9 in Fig 7 2 in the Scanner Control window The DIGE File Naming Format window appears DIGE File Naming Format x Image Group Name Fen model Gel F ds m Standard Scan Dye Image 1 GelF STANDARD Cy2 G 2 Image 2 aF es C Cy3 Image 3 rg Cy5 Image 4 SSE TT E C NONE All images have gel extension OK Cancel e If asingle scan setting has been chosen e g for a Deep Purple stained gel then the resulting output on scan completion will be a filename gel file in the selected folder 7 i e f two or more scan parameter settings were chosen e g for a Cy2 Cy3 Cy5 gel then the resulting output upon sc
10. can be performed in the BVA module it is also possible to perform the following analyses in EDA e Principal Component Analysis Produces an overview of the data Can be used to find outliers in the data e Pattern analysis Finds patterns in expression data e g proteins and spot maps with similar expression profiles Discriminant analysis Finds proteins that discriminate between different samples to find biological markers creates classifiers and assigns samples to known classes depending on expression profiles e g tumor typing e Interpretation Finds the biological context of proteins by integrating biological information and context from in house or public databases It can be used to determine in what pathways and processes a protein is involved the function of the protein etc Ettan DIGE System User Manual 18 1173 17 Edition AB Image analysis 8 8 2 ImageMaster 2D Platinum software 8 2 1 Structure ImageMaster 2D Platinum ensures fast and reliable image comparisons It easily manages multiple image analyses and offers the possibility to automate detection and matching steps with numerous interactive tools for optimizing and manipulating data Furthermore it is possible to attain a higher level of quantitative and qualitative analysis using the robust and sophisticated techniques provided in the application ImageMaster integrates filtering querying reporting and statistical and graphing options so that you can
11. cell wash solution As an alternative 0 5 X phosphate buffered saline PBS can be used However if PBS is used it must be completely removed since it may cause issues with high salt load during electrophoresis Any other wash solutions should be tested for compatibility with the labeling step in controlled experiments The cell wash solution used should not lyse the cells but it should dilute and remove any growth media or reagents that might affect CyDye DIGE Fluor dye labeling process Cell lysis solution It is recommended to use the cell lysis solution Alternatively buffers such as Tris or Bicarbonate can be used in the protein solution The solution should be at a concentration of approximately 30 mM Higher concentration may affect isoelectric focusing Note Ensure that the pH remains between pH 8 0 9 0 in the cell lysate by adding the cell lysis solution Failure to include a suitable buffer will mean that the pH of the solution may fall below pH 8 0 resulting in little or no protein labeling Ettan DIGE System User Manual 18 1173 17 Edition AB Sample preparation and labeling 4 Note The cell lysis solution should not contain any primary amines such as 4 3 2 ampholutes or thiols e g DTT as these will compete with the proteins for CyDye DIGE Fluor dyes resulting in fewer dye labeled proteins This decrease in labeling efficiency may affect the data after scanning and spot detection Protocol The example of protoc
12. higher PMT voltage The gel image appears black The PMT voltage is too high Rescan with lower PMT voltage Appearance of nonspecific background on gel image Platen contaminated with dye from scanning other gels Clean platen Rescan gel on different part of platen to confirm that background does not move with the gel Bacterial mycoplasma contamination in gel running equipment Bacterial mycoplasma contamination in gel running equipment Appearance of small spots sharp peaks in gel image Dirt and dust on gel plates or platen Clean platen or gel plates Rescan gel on different part of platen to confirm that background does not move with gel Dirt in gel Filter acrylamide DeCyder 2D software gives poor spot boundaries Incorrect scan resolution Rescan gels with resolution set to 100 um Faint ill defined gel images possibly with high background Incorrect focal plane selected Re scan with correct focal plane selected Setting gel orientation and scan resolution 108 Ettan DIGE System User Manual 18 1173 17 Edition AB Index Index A Analytical workflow B Batch Processor Cc Cleaning glass plates Cropping CyDye DIGE Fluor dyes recONnSstitutioN anai stability of stock dye solution when to use which dye s s s Working SOLUTION scsccsecissctessecsosssozsccoyandtssgnsannsssssosbesnsseaceccbcgnsecsenej
13. is unable to recognize where individual gels will be located therefore a single file output will be generated Manual cropping is required following the scan Define the number of gels to be scanned Use the drop down menu in the Tray set up area labeled 1 in Fig 7 2 In the DIGE Ettan DALT mode up to two gels can be selected 2 Use the drop down menu under the Acquisition Mode heading labeled 2 in Fig 7 2 to select fluorescence as acquisitor mode Note Scan modes other than fluorescence are available these are covered in the Typhoon User Guide 3 Setup fluorescence scan parameters a Click the Setup button Labeled 3 in Fig 7 2 to activate the Fluorescence Setup window Fluorescence Setup for Typhoon9410 p Link With Use Image Emission Filter PMT Laser Sensitivity M 1 520 BP 40 CY2Blue FAM z 550 Blue2 488 j Noma I 2 580 BP 30 Cy3 TAMRA AlexaFluor546 550 Green 532 XJ Noma F MV 3 ever 30Cy5 550 Red 633 Noma m 4 520 BP 40cv2Btue FAM 60 Normal Y Channel Data Warning If non optimal filter laser or beamsplitter combinations are selected a warning is displayed before scanning is initiated T Disable Warming oven b Select the number of scan channels to be programmed for the sample on the platen Between one and four channels can be programmed To select or deselect scan channels click the Use check box Ettan DIGE System User Manual 18 11
14. labeled lysate on a 1D SDS PAGE gel along with a control lysate already known to label successfully The gel is then scanned at the appropriate wavelength for the relevant CyDye DIGE Fluor dyes The total fluorescence of each labeled sample is then compared The method should also be used to test protein lysates that contain previously untested chemical components Protocol 1 Label 50 ug of the new protein sample with 400 pmol of CyDye DIGE Fluor Cy5 minimal dye 2 Adda volume of each labeled protein lysate equivalent to 50 ug to a microfuge tube 3 Add an equal volume of the 2x gel loading buffer to the labeled protein lysate 4 Heat the samples at 95 C for 5 min to ensure full reduction of the proteins 5 Make a serial dilution of each of the lysates in the 2x gel loading buffer e g 25 ug 12 5 ug and 6 25 ug 6 Make a 12 5 SDS PAGE gel using low fluorescence glass plates The gel should be made with wells into which the samples will be loaded 7 Load each protein serial dilution in successive lanes on the gel 8 Runthesamples until the Bromophenol Blue dye front has nearly reached the bottom of the gel 9 Thoroughly clean the outside of the glass plates with double distilled water 10 Scan the gel at the appropriate wavelength with Typhoon Variable Mode Imager or Ettan DIGE Imager Ettan DIGE System User Manual 18 1173 17 Edition AB 91 A Testing cell lysates for successful labeling A 1 Testing new cell lysate for s
15. lysis buffer Clean sample by precipitation with Ettan 2D Clean Up Kit or increase the amount of dye in the labeling reaction Incorrect concentration of protein in lysate Use a detergent or thiourea compatible protein assay kit e g Protein Determination Reagent or Ettan 2D Quant Kit Check quality of DMF Should be gt 99 8 anhydrous DMF from a bottle that has not been open for longer than 3 months Degraded dye due to hydrolysis of NHS ester or photodegradation of fluorophore Ensure that the appropriate storage conditions have been used for the CyDye DIGE Fluor minimal dye in dark at 20 C Check specific batch expiry date and reconstitution date Incorrect dye protein ratio used 400 pmol of dye per 50 ug of protein is recommended If there is a large concentration of other components which can react with the dye then more dye up to 2 nmol per 50 ug of protein can be used Low pH prior to labeling Check pH is 8 5 immediately prior to labeling If necessary increase pH using higher pH lysis buffer containing 30 mM Tris pH 9 0 10 0 or use 50 mM NaOH 104 Ettan DIGE System User Manual 18 1173 17 Edition AB Trouble shooting guide D D 2 First dimension electrophoresis Problem Cause Remedy Current is zero or too low External electrode contacts are poor Ensure that the electrodes at the bottom of the strip holder one at each end make metal to metal contact
16. protein spot has its own internal standard is the only way to remove gel to gel variation thereby significantly increasing accuracy and reproducibility To capitalize on this ability to multiplex DeCyder 2D software and ImageMaster 2D Platinum software have been specifically designed for the Ettan DIGE system to accurately measure very small protein differences with high confidence DeCyder 2D software and ImageMaster 2D Platinum software contains proprietary algorithms that perform co detection of differently labeled samples within the same gel 1 2 Workflows in 2D analysis Two main workflows using Ettan DIGE System are presented e Analytical workflow e Preparative workflow 1 Introduction 1 2 Workflows in 2D analysis 1 2 1 Analytical workflow The analytical workflow is used to find proteins of interest in an experiment It includes defining the experimental design preparing the samples performing 2D separation of the proteins scanning the gels and analyzing the gel images to find proteins of interest Fig 1 1 outlines an overview of the Ettan DIGE System analytical workflow See Chapters 4 8 for detailed information about the different steps in the analytical workflow 1 Set up the experimental design Gel Cy2 Cy3 Cy5 2 Prepare the samples Unlabeled Cy2 Cy3 Cy5 vy yq vv wW RE Pool portion ne Wil y Labeling y Internal standard 3 Perform first and second dime
17. proteins from re oxidizing Formation of charge trains Protein carbamulation Check that all solutions containing urea were prepared freshly and ensure that all solutions containing urea were not heated above 37 C at any time Horizontal streaking or incomplet ely focused spots High sample load Reduce sample load by adding less sample to the rehydration solution Insufficient focusing time Increase total Vh for focusing 106 Ettan DIGE System User Manual 18 1173 17 Edition AB Trouble shooting guide D Problem Cause Remedy Vertical streaking or incomplet ely focused spots SDS depletion during second dimension electrophoresis Use 0 2 SDS in the running buffer for both top and bottom buffer tanks Ensure that the gel has been prepared with the correct concentration of SDS Over alkylated proteins Use lower pH higher DTT concentration or lower iodoacetamide concentration when equilibrating Immobiline DryStrips Sample insolubility and particulates Insufficient chaotrope or detergent Ensure the correct solubilization solution has been used Cloudy samples Remove insoluble material from the sample using ultracentrifugation Poor first dimension focusing lonic detergent concentration too high in lysis buffer If SDS is used in sample preparation the final concentration must not exceed 0 25 after dilution into the rehydration solution Als
18. reference markers on the gel backing short glass plate Ettan DIGE System User Manual 18 1173 17 Edition AB 83 9 Preparative workflow 9 5 Post staining with Deep Purple Total Protein Stain 84 9 4 3 Second dimension SDS PAGE We recommend using the Ettan DALT electrophoresis system The orientations of the Immobiline DryStrips and the reference markers are critical to ensure that the picking gel is easily matched to the analytical gels during image analysis and that the correct spots are picked from the gel when the pick list is exported to the Ettan Spot Picker 1 After a picking gel has been poured containing suitable reference markers position the gel so the front of the reference markers are facing you 2 Equilibrate the Immobiline DryStrips as described in Section 6 4 Equilibration of focused Immobiline DryStrips 3 Load the equilibrated Immobiline DryStrips see Section 6 5 Loading of focused Immobiline DryStrips ensuring that the acidic pointed end of the Immobiline DryStrips is on the left hand side of the gel as shown in Fig 9 2 4 Torun the gel see 6 6 for further information 9 5 Post staining with Deep Purple Total Protein Stain Note Ifthe sample used has been labeled with CyDye DIGE Fluor saturation dye post staining of the second dimension SDS PAGE gel is not required The fluorescent Deep Purple Total Protein Stain fits into the standard 2D gel electrophoresis workflow and is particularly suitable for
19. special protocols for DIGE system preparative workflow The four appendices cover other important information Ettan DIGE System User Manual 18 1173 17 Edition AB 11 1 Introduction 1 4 Ettan DIGE System related products manuals 1 4 Ettan DIGE System related products manuals Because Ettan DIGE System comprises many different products references to the appropriate manuals for further details are made when necessary see Table 1 1 The manuals are also available on the web www ettandige com Table 1 1 Ettan DIGE System related products manuals Manual Code no Ettan IPGphor 3 Control Software User Manual 11 0034 59 Ettan IPGphor 3 Instrument User Manual 11 0034 58 Ettan IPGphor Cup Loading Manifold User Manual 11 0034 60 2D Electrophoresis Principles and Methods 80 6429 60 Ettan DALTtwelve system User Manual 80 6476 53 Ettan DALTsix system User Manual 80 6492 49 Typhoon User s Guide 63 0028 31 Ettan DIGE Imager User Manual 11 0036 59 mageQuant TL User Manual 63 0050 82 DeCyder 2D Software User Manual 28 4010 06 DeCyder Extended Data Analysis Module User Manual 28 4010 07 mageMaster 2D Platinum 6 0 User Manual 11 0034 38 Deep Purple Total Protein Stain Instructions RPN6305PL Ettan Spot Picker User Manual 18 1147 61 Ettan Digester User Manual 18 1167 31 Ettan Spotter User Manual 11 0003 41 Ettan Spot Handling Workstation User Manual 18 1153 55 12 Ettan DIGE System User Manual 18 1173 17 Edition AB DIGE concepts 2
20. system is capable of detecting and quantifying differences as small as 10 between samples above system variation with greater than 95 statistical confidence 2 2 CyDye DIGE Fluor dyes Ettan DIGE system is based upon the specific properties of the CyDye DIGE Fluor dyes There are two different CyDye DIGE Fluor dyes available CyDye DIGE Fluor minimal dyes and CyDye DIGE Fluor saturation dyes The saturation dyes are also known as labeling kit for scarce samples The key differences between CyDye DIGE Fluor minimal and saturation dyes are summarized in Table 2 1 e Use CyDye DIGE Fluor minimal dyes for normal applications Multiplexing up to three samples and labeling of 50 ug protein is possible e Use CyDye DIGE Fluor saturation dyes when samples are precious or available in very small amounts Multiplexing up to two samples and labeling of 5 ug protein is possible Ettan DIGE System User Manual 18 1173 17 Edition AB DIGE concepts 2 Table 2 1 Summary of key differences between CyDye DIGE Fluor minimal and saturation dyes Saturation labeling Minimal labeling Sample preparation Cell lysis buffer is at pH 8 0 Cell lysis buffer is at pH 8 5 Dyes aleimide dyes Label cysteine residues Two dyes available Reconstituted stock at 2 mM analytical gels or 20 mM preparative gels o dilution of stock solution NHS ester dyes Label lysine residues Three dyes available Reconstituted stock at 1 mM Wor
21. that can be directly exported to Ettan Spot Picker or Ettan Spot Handling Workstation 3 Protein spots of interest are then automatically picked from the gel by Ettan Spot Picker or Ettan Spot Handling Workstation This chapter provides information of changes to the protocols described in chapters 4 8 one must make to ensure an efficient preparative workflow The workflow is outlined in Fig 9 1 9 1 1 Staining of preparative gels Depending on which CyDye DIGE Fluor dye has been used for sample labeling there are two options for visualization of preparative gels e For CyDye DIGE Fluor minimal dyes the second dimension SDS PAGE preparative gels must be post stained Use fluorescent protein stains such as Deep Purple for post staining Note CyDye labeled proteins are approximately 450 Da larger in molecular weight compared to non labeled proteins e For CyDye DIGE Fluor saturation dyes no post staining is required Use the preparative dye vial provided in the kit instead This vial provides amounts of CyDye DIGE Fluor saturation dyes enough for labeling preparative samples Ettan DIGE System User Manual 18 1173 17 Edition AB 79 9 Preparative workflow 9 1 Introduction Sample preparation and labeling Unlabeled Cy3 Alternative 1 Unlabeled sample post staining required y Protein extraction y Cells Sample Alternative 2 Labeled sample no post staining required R Labeling with CyDye DIGE Fluor y P
22. the labeling efficiency by removal of endogenous small molecules It also may improve spot resolution and increase the number of spots detected Ettan DIGE System User Manual 18 1173 17 Edition AB 31 4 Sample preparation and labeling 4 4 pH adjustment 32 11 4 4 Check the pH of the cell lysate and adjust if necessary see section 4 4 The cell lysate can now be stored in aliquots at 70 C until protein yield is to be determined Note When using Ettan 2D Clean Up Kit the pH drops considerably and adjustment of pH to 8 9 is required pH adjustment Check the pH of the cell lysate protein sample by spotting a small volume 1 3 ull on a pH indicator strip Optimal cell lysate pH is 8 5 for CyDye DIGE Fluor minimal dyes and 8 0 for CyDye DIGE Fluor saturation dyes Note The use of cell lysates with optimal pH as described above is very important A lower pH than optimal will make labeling ineffective and a higher pH than optimal will make the labeling unspecific Adjust the pH if the pH of the cell lysate is outside the desired range Normally the pH needs to be increased To increase the pH in the cell lysate make some more cell lysis solution with tris base without the protein with pH 9 5 or higher Add aliquots of the new lysis solution to the cell lysate This will gradually increase the pH of the cell lysate Stop when the pH of the protein sample is at pH 8 5 for minimal labeling Alternatively the
23. the suspension at 800 x g and 4 C for 2 minutes 5 Remove the supernatant and resuspend a cell pellet containing 5 10 x 10 cells in 200 ul cold labeling buffer HBSS pH 8 5 1M Urea Add 1 5 ul CyDye working solution 600 pmoles Mix briefly by vortexing at slow speed 6 Label the cell surface for 20 minutes on ice in darkness Then add 20 ul of 20 mM lysine and incubate another 10 minutes Ettan DIGE System User Manual 18 1173 17 Edition AB 97 B Labeling of cell surface proteins B 1 Selective labeling of cell surface proteins 98 After labeling and quenching wash the cells twice in 500 ul cold HBSS pH 7 4 by centrifugation at 800 x g and 4 C for two minutes Resuspend the pellets directly in 150 ul cold lysis buffer 7M Urea 2M Thiourea 4 CHAPS 30 mM Tris 5 mM MgAc pH 8 5 and leave on ice for at least 1 hour with occasional vortexing Centrifuge the lysate at 10 000 x g and 4 C for 5 minutes Transfer the supernatant to a new tube Note Fractionation of the sample may be desired It is not necessary for improved detection of cell surface proteins but could be used to verify lack of labeling of cytosolic proteins Then resuspend the cell pellet in 150 ul cell lysis buffer included in the membrane fractionation kit 2D Sample Prep for Membrane Proteins Pierce After fractionation the sample can be directly applied on 2D electrophoresis Add 2x Sample buffer to the lysed cell surface labeled sample Now the sam
24. use with the Ettan DIGE system The recommended workflow involves the matching of Deep Purple post stained preparative gels with CyDye labeled analytical gels Deep Purple has been shown to be compatible with the image analysis softwares and the stain is compatible with manual or automated spot picking and mass spectrometry for protein identification applications 1 Place an appropriate volume of fixation solution into the containers that will be used to process gels The recommended volume of fixation solution required is 20 fold excess of the gel volume 1000 ml for Ettan DALT gels 2 Dismantle the electrophoresis apparatus Remove one glass plate and place the gel attached to the glass plate into fix solution Note Place only one gel in each container The Immobiline DryStrip can be left attached to help with gel orientation Ettan DIGE System User Manual 18 1173 17 Edition AB Preparative workflow 9 3 Incubate in the fixation solution overnight at room temperature with gentle agitation 4 Take the stain out of the 15 C to 30 C freezer and allow to stand at room temperature for 5 10 minutes 5 Pour off the fixation solution and replace with the wash solution 1000 ml for Ettan DALT gels Wash with gentle agitation for 30 minutes 6 Pour off the wash solution and replace with 500 ml water To make up the working stain solution briefly shake the stain concentrate and add 2 5 ml Deep Purple to make a 1 200 dilution Cover th
25. with the appropriate electrode area Internal electrode contacts are poor Ensure that the gel makes contact with both electrodes in the strip holder Immobiline DryStrips not fully rehydrated Check that the Immobiline DryStrips are fully rehydrated along their entire length Electrical contact at the electrodes is reduced by incomplete rehydration No conduction through electrode wicks Check that the electrode wicks if used were moistened prior to use Voltage too low or does not reach the maximum set value Current limit setting is incorrect Check that the current limit is properly set Incorrect number of Immobiline DryStrips Check that the correct number of strips in place is set on the IPGphor program No Pharmalytes added to sample The recommended Pharmalyte concentration is 1 0 v v Sparking or burning in strips High current Do not exceed the recommended setting of 50 uA per Immobiline DryStrip Strips not rehydrated Check that Immobiline DryStrips are fully rehydrated along their entire length Presence of bubbles Check that any large bubbles trapped under the Immobiline DryStrips after wetting with rehydration solution are removed prior to focusing Strips drying out during focusing Ensure that sufficient Immobiline DryStrips Cover Fluid has been applied High salt concentration Clean sample to remove excess salts Proteins have no
26. 0 difference between samples can be made with more than 95 statistical confidence Tip Ensure DIGE images are cropped before being loaded into DeCyder 2D 8 1 DeCyder 2D software DeCyder 2D software is specially designed for Ettan DIGE system use It enables the production of quantitative data of unparalleled accuracy supported by statistical tests This gives confidence that the results achieved reflect true biological outcomes and are not due to the system DeCyder 2D is a fully automated image analysis software suite for detection quantitation positional matching and differential protein abundance analysis An optional add on module Extended Data Analysis EDA can handle up to 1000 spot maps The raw data gel images are linked to EDA and multivariate analysis of data can be opened for display via the BVA module This section briefly outlines the features and capabilities of the software For a detailed guide please refer to the DeCyder 2D Software User Manual and DeCyder Extended Data Analysis module User Manual which both include a series of tutorials designed to provide new users with the means to gain a rapid understanding of the software s capabilities Online helps are also integrated with the software providing help for the different parts of the software 8 1 1 Modules DeCyder 2D version 6 5 software comprises six modules where the EDA module is optional Image Loader Import of scanned gel images into a project with
27. 2 DIGE concepts 2 1 Introduction 2 1 1 2D analysis result variation 2D analysis experiments commonly address questions like protein level differences caused by a disease state drug treatment life cycle stage etc Some protein level differences studied are small and the results are affected by experimental variation originating both from the system and from inherent biological variation System related result variation System related result variation may arise for two reasons 1 Gel to gel variation which can result from differences in electrophoretic conditions between first dimension strips or second dimension gels gel distortions sample application variation and user to user variation 2 Variation due to user specific editing and interpretation when using the data analysis software Inherent biological variation Inherent biological variation arises from intrinsic differences that occur within a population For example differences from animal to animal plant to plant or culture to culture which have been subjected to identical conditions 2 1 2 Improvement of results by use of Ettan DIGE system There are three main factors enabling Ettan DIGE System to provide greater accuracy than conventional 2D analysis 1 Multiplexing that is to run multiple samples on the same gel 2 Use of an internal standard for all proteins which can be run on all gels in a set of experiments 3 Experimental designs unique to this technique se
28. 2 Define Region of Interest Ctrl R 2 Crop using Edit Crop Dataset or the crop current dataset button on the toolbar 3 For image analysis save the cropped images by selecting File Export Gel Files from Dataset to Folder This method only saves the name gel files and allows images from multiple gels to be saved in a common folder 4 To retain dataset functionality save the cropped images by selecting File Save As Ettan DIGE System User Manual 18 1173 17 Edition AB 71 7 Image acquisition 7 6 Cropping using ImageQuant TL 7 6 1 ImageQuant TL trouble shooting If out of memory messages appear while using ImageQuant TL causing the software to crash this may be corrected by decreasing the virtual memory under 1Gb Remember to change the setting again when using the image analysis softwares which need a large virtual memory to function correctly To set the size of the virtual memory 1 Right click on My computer and select Properties 2 Select the Advanced tab 3 In the Performance area click the Settings button 4 Inthe Performance area click the Settings button 5 Click Change to change the virtual memory to the appropriate value and click OK to change the setting 6 Restart the computer to apply the new setting 72 Ettan DIGE System User Manual 18 1173 17 Edition AB Image acquisition 7 7 7 Ettan DIGE Imager Ettan DIGE Imager is a scanning CCD camera based instrument designed for high resolution i
29. 3 17 Edition AB Sample preparation and labeling 4 Note Check that the dye solution has an intense color During transport the dye powder may spread around the inside surface of the tube including the lid If the dye has not an intense color then pipette the solution around the tube and lid to ensure resuspension of dye Vortex and spin down For dilution of the CyDye DIGE Fluor minimal dye stock solution 1 Dilute the minimal dye stock solution to working dye solution concentration see section 4 7 2 2 The dye stock solution should be stored in a light excluding container at 20 C Return to freezer as soon as possible after use The dye stock solution is stable for up to three months at 20 C 4 7 2 Preparation of working dye solution Amount of dye required for labeling It is recommended that 400 pmol of dye is used to label 50 ug of protein If labeling more than 50 ug of protein then the dye protein ratio must be maintained for all samples in the same experiment Other dye protein ratios can be used but must be optimized for the sample by testing the labeling on an SDS PAGE gel see Appendix A Dilution of dye stock solution Prior to labeling the dye stock solution is diluted with DMF see above to a working dye solution A concentration of 400 pmol l is recommended Note The working dye solution is only stable for one week at 20 C 1 Briefly spin down the dye stock solution in a microcentrifuge 2 Add 2 ul dye s
30. 4 5 ug sample C4 6 5 ug 4 17ug each of A1 4 B1 4 C1 4 5 ug sample B2 7 5 ug 4 17ug each of A1 4 B1 4 C1 4 5 ug sample A1 8 5 ug 4 17ug each of A1 4 B1 4 C1 4 5 ug sample C3 9 5 ug 4 17ug each of A1 4 B1 4 C1 4 5 ug sample B1 10 5 ug 4 17ug each of A1 4 B1 4 C1 4 5 ug sample A3 11 5 ug 4 17ug each of A1 4 B1 4 C1 4 5 ug sample C1 12 5 ug 4 17ug each of A1 4 B1 4 C1 4 5 ug sample B3 Total gels 12 Note It is possible to use CyDye DIGE Fluor minimal dyes in the same manner labeling only with two dyes 28 Ettan DIGE System User Manual 18 1173 17 Edition AB Sample preparation and labeling 4 4 Sample preparation and labeling 4 1 Introduction The preparation of a cell lysate and subsequent labeling with CyDye DIGE Fluor minimal dyes or CyDye DIGE Fluor saturation dyes is detailed in this chapter The reagents and conditions stated here are those which have been found to be the most consistently useful across many sample types However there will be cases where some individual optimization of lysis conditions is required See Section 2 2 for information of CyDye DIGE Fluor minimal dyes and CyDye DIGE Fluor saturation dyes and Chapter 9 for special changes in protocols when running preparative second dimension SDS PAGE for spot picking of proteins of interest Sample preparation for analytical gels Unlabeled Cy2 Cy3 Cys yuyu eS Cells Protein extraction Labeling wit
31. 61 7 Image acquisition 7 2 Workflow 7 2 Workflow The workflow for scanning gels with Typhoon Variable Mode Imager and cropping gel image files consist of following steps 1 2 7 3 Cleaning Typhoon Placing the gels in Typhoon Selecting scan parameters Scanning of the gels Cropping of image files Cleaning Typhoon The following cleaning procedure has been shown to be compatible with the Typhoon Variable Mode Imager to remove contamination caused by fluorescent products 7 4 Wipe the glass platen with 10 H20 hydrogen peroxide using lint free paper such as Crew Wipers Rinse the platen with high purity water A pre scan can be done to check for contaminants that may affect results of scans Placing gels in Typhoon Note Wear powder free gloves The powder used in laboratory gloves can 62 fluoresce and may also scatter light affecting image quality Turn on the Typhoon Variable Mode Imager and leave the instrument to warm up for at least 30 min prior to scanning Once the instrument is warmed up it will display READY status Ensure that the gel glass plates are clean dry and free from lint Tip For applications using Ettan DIGE system the recommended glass plates have low fluorescence characteristics The gel can then be scanned still assembled within the plates Manipulation will be easier and there will be less risk of damage to the gel Ettan DIGE System User Manual 18 1173 17 Edition AB I
32. 73 17 Edition AB 65 7 Image acquisition 7 5 Scan parameters and scanning c Select appropriate emission filters from the Emission Filter list This list displays the filters that are installed on the Typhoon Variable Mode Imager along with a description of the typical filter use The Scanner Control software automatically suggests the laser to use with the emission filter selected See Table 7 1 for recommended emission filters and laser combinations The CyDye DIGE Fluor dye filter and laser combinations are selected to give the optimum results with minimal cross talk Table 7 1 Recommended emission filters and laser combinations Dye Emission Filter nm Laser Cy2 520 BP 40 Blue2 488 Cy3 580 BP 30 Green 532 Cy5 670 BP 30 Red 633 Deep Purple 560 LP Green 532 Tip For Deep Purple post stained analytical gels it is recommended to use a 457 nm laser excitation in conjunction with a 610 nm band pass emission filter or equivalent if not using a Typhoon scanner This will minimize any potential cross talk between Deep Purple and the CyDye DIGE fluor dyes d Set the PMT voltage for each scan wavelength A quick pre scan at 500 or 1 000 um pixel resolution should be performed initially to identify a suitable voltage For further details see section 7 5 1 e Select normal as Sensitivity scan setting for each scan wavelength usually sufficiently sensitive for 2D DIGE applications f Select the Sensitivi
33. Clip down the electrodes firmly onto the electrode papers Ensure that there is good contact between the paper electrodes and the metal Cup loading protocol only Clip a loading cup onto the acidic end of the strip so it is positioned between the two electrodes The cup should form a good seal with the Immobiline DryStrips Note To check for a good seal fill the cup to the top with PlusOne DryStrips Cover Fluid Observe the level of the fluid to check if it is decreasing If a leak is detected remove the PlusOne DryStrips Cover Fluid and reposition the sample cup Apply 108 ml of PlusOne DryStrips Cover Fluid allowing the oil to spread so it completely covers the Immobiline DryStrips even if fewer than twelve strips will be loaded in the Manifold Cup loading protocol only Add samples prepared as described in section 4 8 Up to 100 ul of a protein sample can be loaded into the bottom of the sample cup Close the lid of Ettan IPGphor 3 instrument Now it is possible to start the first dimension isoelectric focusing see section 5 4 46 Ettan DIGE System User Manual 18 1173 17 Edition AB First dimension isoelectric focusing IEF 5 5 4 First dimension isoelectric focusing IEF First dimension isoelectric focusing using Ettan IPGphor 3 instrument can be controlled by either using a PC software Ettan IPGphor 3 Control software or the embedded instrument software Use of Ettan IPGphor 3 Control software is recommended for eas
34. GE Healthcare Ettan DIGE System User Manual Ettan Important user information All users must read this entire user manual to fully understand the safe use of Ettan DIGE System For safe use of Ettan DIGE System related products described in this user manual see corresponding manuals WARNING The WARNING sign highlights instructions that A must be followed to avoid personal injury It is important not to proceed until all stated conditions are met and clearly understood CAUTION The Caution sign highlights instructions that must be followed to avoid damage to the product or other equipment It is important not to proceed until all stated conditions are met and clearly understood Note The Note sign is used to indicate information important for trouble free and optimal use of the product Contents 1 Introduction 1 1 Ettan DIGE System Overview uumeemmmsmnssssoesssssonssssoorssssnersssserrnssennnnannnn nan 9 1 2 Workflows in 2D analysis 1 2 1 Analytical WOrKFIOW ssssssssssssssssssssssssssssscccssesscssecceseecessessssssssssssssssnunusssssseesees 1 2 2 Preparative workflOW essssssssssssssssssccsesscssscesceccesssssssssssssnnussessessscesceseeeeeeseeseees 1 3 Ettan DIGE System User Manual mmm 11 1 4 Ettan DIGE System related products manuals 12 2 DIGE concepts 21 WIOCUCTION ceeceececessssssssssssesssssceecsscecssseceesscecsseceesscceseseeseesecesssecesssee
35. H 8 0 l E 0 o S Maleimide reactive group Fig 2 2 Schematic of the saturation labeling reaction 0 Ettan DIGE System User Manual 18 1173 17 Edition AB 17 2 DIGE concepts 2 3 The internal standard With CyDye DIGE Fluor saturation dyes 5 ug protein is labeled in each reaction The dye is added to the protein under such conditions that all available cysteine residues of the proteins are labeled For that reason this type of labeling has been called saturation labeling To achieve optimum labeling a high dye to protein ratio is required 2 3 The internal standard The multiplexing capability of the 2D DIGE methodology enables the incorporation of the same internal standard on every second dimension SDS PAGE gel The internal standard is a pool of all the samples within the experiment and therefore contains every protein from every sample The internal standard is used to match and normalize the protein patterns across different gels thereby negating the problem of inter gel variation a common problem in standard one sample per gel 2D electrophoresis experiments The internal standard allows accurate quantitation of differences between samples with an associated statistical significance Quantitative comparisons of protein between samples are made on the relative change of each protein spot to its own in gel internal standard It enables accurate statistical quantification of induced biological change between samples
36. IEF msmsmsmsmmsme 47 5 5 RECIPES N E E d2ictess ntessarbteniadsiv E 48 Second dimension SDS PAGE 6 1 Ettan DALT electrophoresis SYStem wiccecceccccssssssssssssessesssssssessssssseueees 51 E OTTO AAE E A AN E ANE EEE E 52 6 3 CASTING homogeneous GEIS on eesssssssssssesnsssssssccsssssssssssusesessecsecsessssssesnseesses 52 6 4 Equilibration of focused Immobiline DryStrips 54 6 5 Loading of focused Immobiline DryStrips 55 6 6 Second dimension SDS PAGE se 56 6 7 REGU DSS A E ET EE ean ctestamndsaeuesamess 58 Image acquisition 7 1 Typhoon Variable Mode Imager Z 2 gt WOTKFIOW sssseicecetiecaiancasaniees i ES Cleaning TYPNOON sesisnpaanianinmuneena nan AEN LA Placinggels in TPN ONiiesesssnsimunsenniaisanunnan 7 5 Scan parameters and scanning 7 5 1 Pre scanning to identify a suitable PMT voltage 7 6 Cropping using ImageQuant TL ou eeeccsseeesseseeeees wll 7 6 1 ImageQuant TL trouble SAOOtiN Os 72 1 1 Etta DIGE IMIOGER ssssinsisistaneal inredni brinner 73 Image analysis 8 1 DESY GERAD SOWIE eeann iA 75 8 1 1 MOCO Sese o a e geia js e 75 8 2 ImageMaster 2D Platinum software omsmeeeeccumssssmsmsssmsmmsmsmmm 77 8 2 1 Structure mas 8 2 2 Image analysis WOFKFIOW sssssssssssssssessssssscscccsecsesccseceecsessessensnssssssnsnasssnnenteees 77 Ettan DIGE System User Manual 18 1173 17 Edition AB Contents 9 Preparative workflow 91 gt TNE OC OCT N assins 9 1 1 Staining of prepa
37. O and NazCOz in 750 ml water Make up to 1 000 ml with distilled water The pH of the solution should be pH 10 11 and should be verified 20 fold excess of the gel volume should be used This solution can be stored for up to 2 weeks 88 Ettan DIGE System User Manual 18 1173 17 Edition AB Preparative workflow 9 Working stain solution Reagent Quantity Final concentration Deep Purple 2 5 ml 1 200 dilution Make up to 500 ml with distilled water 10 fold excess of the gel volume should be used This solution should be made fresh at the time of use by adding an appropriate aliquot of Deep Purple to water in the gel staining tank If necessary it is possible to store this solution protected from exposure to light for up to 1 week at 2 8 C or 24 h at room temperature Storage solution Reagent Quantity Final concentration Glacial acetic acid 75 ml 7 5 v v Make up to 1 000 ml with distilled water Ettan DIGE System User Manual 18 1173 17 Edition AB 89 9 Preparative workflow 9 10 Recipes 90 Ettan DIGE System User Manual 18 1173 17 Edition AB Appendix A Testing cell lysates for successful labeling A Testing cell lysates for successful labeling A 1 Testing new cell lysate for successful labeling It is important to check that labeling of the proteins is optimized before the samples are taken through the 2D electrophoresis process The method involves running a small sample of the freshly
38. S PAGE gel thus making multiplexing possible Sensitivity The dyes afford great sensitivity down to 25 pg of a single protein and a linear response to protein concentration up to five orders of magnitude 10 In Ettan DIGE System User Manual 18 1173 17 Edition AB 15 2 DIGE concepts 2 2 CyDye DIGE Fluor dyes 16 comparison silver stain detects 1 60 ng of protein with a dynamic range of less than two orders of magnitude Protein labeling CyDye DIGE Fluor minimal dyes have an NHS ester reactive group and are designed to form a covalent bond with the epsilon amino group of lysine residues in proteins via an amide linkage The dye is added to the protein such that the amount of dye is limiting within the labeling reaction The lysine amino acid in proteins carries an intrinsic 1 charge at neutral or acidic pH CyDye DIGE Fluor minimal dyes also carry a 1 charge which when coupled to the lysine replaces the lysine s 1 charge with its own ensuring that the pl of the protein does not significantly alter With CyDye DIGE Fluor minimal dyes 50 ug protein is labeled in each reaction The ratio used ensures that the dyes label approximately 1 2 of lysine residues so each labeled protein carries only one dye label and is visualised as a single protein spot The CyDye DIGE Fluor minimal dyes therefore only label a small proportion of the total protein in a sample For that reason this type of labeling has been called minimal la
39. The 2D DIGE methodology is the only technique to enable accurate standardized quantitation 2 3 1 Advantages of using an internal standard The recommended protocol for experiments with more than three samples includes an internal standard that is run on all gels within an experiment together with up to two different labeled protein samples Linking every sample in all gels to a common internal standard offers a number of advantages e Accurate quantification and accurate spot statistics between gels e Increased confidence in matching between gels e Flexibility of statistical analysis depending on the relationship between samples e Separation of induced biological change from system variation and inherent biological variation 18 Ettan DIGE System User Manual 18 1173 17 Edition AB DIGE concepts 2 2 3 2 Examples of benefits using an internal standard The examples below illustrate the benefits of an internal standard Fig 2 3 shows the theoretical scan results of two gels Each gel contained two protein samples labeled with CyDye DIGE Fluor Cy3 or Cy5 minimal dyes and the same pooled internal standard sample labeled with CyDye DIGE Fluor Cy2 minimal dye If the gels illustrated in Fig 2 3 were analyzed without an internal standard the conclusion would be that the volume of the highlighted protein spot in samples 1 and 2 has remained the same but is increased slightly in sample 3 and further in sample 4 However reference to the
40. acing gels scan parameters scanning workflow X XML Toolbox Ettan DIGE System User Manual 18 1173 17 Edition AB 111 Index 112 Ettan DIGE System User Manual 18 1173 17 Edition AB www gehealthcare com GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden Cy CyDye DeCyder Ettan Ettan Dige Imager Deep Purple Immobiline ImageMaster ImageQuant TL IPGphor PlusOne and Typhoon are trademarks of GE Healthcare companies GE imagination at work and GE monogram are trademarks of General Electric company Deep Purple Total Protein Stain is exclusively licensed to GE Healthcare from FluoroTechnics Pty Ltd Deep Purple Total Protein Stain may only be used for applications in life science research ImageMaster has been has been developed by the Swiss Institute of Bioinformatics in collaboration with GeneBio and GE Healthcare Microsoft Windows Windows XP Word Excel Internet Explorer are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries Pentium is a trademark of Intel Corporation or its subsidiaries Unless otherwise agreed in writing all goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request GE Healthcare reserves the right to make changes in specifications and features shown herein or di
41. al flask and heat on a low setting in the microwave for 1 minute Ensure all the agarose has melted Allow the solution to cool slightly before use Store at room temperature Do not keep for more than 1 month 1 w v Bromophenol blue stock solution Reagent Quantity Final concentration Bromophenol blue 100 mg 1 Tris base 60 mg 50 mM Make up to 10 ml with double distilled water Ettan DIGE System User Manual 18 1173 17 Edition AB Image acquisition 7 7 Image acquisition Typhoon Variable Mode Imager is recommended for scanning of DIGE second dimension SDS PAGE gels It is also possible to use Ettan DIGE Imager see section bls 7 1 Typhoon Variable Mode Imager WARNING It is important that you read the safety instructions in the Typhoon User Guide before you start work Typhoon Variable Mode Imager is fully optimized as part of Ettan DIGE System Fig 7 1 It is optimized to image the CyDye DIGE Fluor dyes characteristics Several models of the Typhoon Variable Mode Imager system are available For Ettan DIGE system use Typhoon Variable Mode Imager Trio 9400 and 9410 are recommended as all three CyDye DIGE Fluor minimal dyes can be detected However seamless integration with DeCyder 2D software is ensured for all models Fig 7 1 Typhoon 9410 Variable Mode Imager For a detailed guide on Typhoon Variable Mode Imager see Typhoon User Guide Ettan DIGE System User Manual 18 1173 17 Edition AB
42. an completion will be a filename ds file in the selected folder and a new folder called filename dir In this new folder will be the user named gel files The filename ds file allows the scanned images to be overlaid in ImageQuant TL whilst the user named gel files are the individual scan channel outputs and can be viewed as separate files 10 The terms STANDARD Cy2 Cy3 and Cy5 are automatically appended to the file These terms are also automatically picked up during image analysis so reducing the requirement for user input 68 Ettan DIGE System User Manual 18 1173 17 Edition AB Image acquisition 7 11 The term STANDARD can be assigned to the file name of the image which contains the internal standard by clicking the relevant check box If not required the STANDARD name can be removed by clicking the NONE check box The filenames can also be manually edited 12 Click the OK button The Multiple Sample Name dialog is displayed Multiple Sample Naming x Sample FileName sre 1 F E coli model Expt 5 Group A1 ds 25 177 MB 2 F AE coli model Expt 5 Group A2 ds 25 177 MB Edit Sample File Name Use Common Setting for All Samples Folder JE coli model Browse Base File Name Expt 5 Group A Browse Set Cancel SCAN 13 Save the images by choosing the folder and filename for each gel individually Click on Edit Sample File Name 14 Entering details using Folder or Base File Name all
43. are Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Asia Pacific Tel 852 2811 8693 Fax 852 2811 5251 Australasia Tel 61 2 9899 0999 Fax 61 2 9899 511 Austria lel 01 5 6U6 1619 Fax 01 5 606 1627 Belgium Tel 0800 73 888 Fax 03 272 1637 Canada Tel 800 463 5800 Fax 800 567 1008 e Central East amp South East Europe Tel 43 1 982 3826 Fax 43 1 985 8327 e Denmark Tel 45 16 2400 Fax 45 16 2424 e Finland amp Baltics Tel 358 0 9 512 39 40 Fax 358 0 9 512 39 439 e France Tel 01 69 35 67 00 Fax 01 69 41 96 77 Germany Tel 0761 4903 490 Fax 0761 4903 405 e Italy Tel 02 27322 1 Fax 02 27302 212 e Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 e Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 Middle East amp Africa Tel 30 210 9600 687 Fax 30 210 9600 693 Netherlands Tel 0165 580 410 Fax 0165 580 401 e Norway Tel 815 65 555 Fax 815 65 666 Portugal Tel 21 417 7035 Fax 21 417 3184 Russia amp other C I S amp N I S Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 South East Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 e Spain Tel 93 594 49 50 Fax 93 594 49 55 e Sweden Tel 018 612 1900 Fax 018 612 1910 Switzerland Tel 0848 8028 12 Fax 0848 8028 13 UK Tel 0800 616928 Fax 0800 616927 USA Tel 800 526 3593 Fax 877 295 8102 imagination at work User Manual 18 1173 17 AB
44. at least 100 um Ettan DIGE System User Manual 18 1173 17 Edition AB 85 9 Preparative workflow 9 7 Matching gels and creating a pick list 86 3 Ensure that both reference markers can be clearly seen and that they appear as circles when the gel image is checked If the markers cannot be seen then re scan the gel adjusting the area to be scanned appropriately 4 When scanning is finished place the gel into the storage solution 7 5 acetic acid at 4 C in the dark 5 Clean the scanner platen after use to remove any fluorescent residue If the platen is not thoroughly cleaned this residue can interfere with subsequent scans producing high background levels See 7 3 for information of how to clean the scanner platen 9 7 Matching gels and creating a pick list The matching of gels and creating a pick list is performed in DeCyder 2D For information on the different steps in the workflow below see DeCyder 2D User Manual and DeCyder Extended Analysis module User Manual 9 8 Spot picking the gel After a pick list has been created from the image analysis software spot picking can be performed The procedure of picking and digesting spots can be performed by manual transfer of gels and microplates between the Ettan Spot Picker Ettan Digester and Ettan Spotter or fully automatically in the integrated Ettan Spot Handling Workstation Processing is done in three main steps 1 Spot picking from second dimension SDS PAGE gels Selecte
45. ating data Exports spot coordinates to a spot excision robot exports gel data to a database for example via XML format or imports experimental information to be included in annotations Reporting results Display information on specific gels and gel components spots matches classes annotations at any moment during the analysis It is possible to display use save customize and edit reports Several specific report types are available Controlling and automating gel analyses As with reporting results operations that were carried out on gels can be checked at any time using the History function It is also possible to create Scripts for automating parts of your analysis A multiple undo redo feature is also available Ettan DIGE System User Manual 18 1173 17 Edition AB Preparative workflow 9 9 Preparative workflow 9 1 Introduction When proteins of interest have been identified in 2D DIGE experiments the corresponding spots can be picked and analyzed in a mass spectrometer for protein identification Although spots of interest can be picked directly from post stained analytical SDS PAGE gels spot picking from preparative scale gels provide more material for analysis by mass spectrometry Ettan DIGE system is fully compatible with mass spectrometry analysis 1 A preparative gel can be matched to previously run analytical gels by DeCyder 2D software 2 DeCyder 2D software will generate a pick list of spots of interest
46. beling lt en M SA 4 pce N Fig 2 1 Schematic of the minimal labeling reaction CyDye DIGE Fluor minimal dye contain ing NHS ester active group covalently binds to the lysine residue of a protein via an amide linkage IN Ettan DIGE System User Manual 18 1173 17 Edition AB DIGE concepts 2 2 2 2 CyDye DIGE Fluor saturation dyes Chemical description CyDye DIGE Fluor saturation dyes also known as labeling kit for scarce samples are two spectrally resolvable dyes Cy3 and Cy5 matched for mass and charge Each CyDye DIGE Fluor saturation dye when coupled to a protein will add approximately 677 Da to the mass of the protein A protein labeled with any of the CyDye DIGE Fluor saturation dyes will migrate to the same position on the second dimension SDS PAGE gel in this way making multiplexing possible Sensitivity The dyes afford great sensitivity with detection lower than 25 pg of a single protein and a linear response to protein concentration up to five orders of magnitude 10 Protein labeling CyDye DIGE Fluor saturation dyes have an maleimide reactive group which is designed to form a covalent bond with the thiol group of cysteine residues in proteins via a thioether linkage CyDye DIGE Fluor saturation dyes have a neutral charge and will not affect the pl of the labeled protein Reduction step s TCEP 1 h 37 C s HS pH 8 0 Coupling step on HH o 37 C 30 min o one p
47. ction quantitation normalization internal standardization and inter gel matching The benefits are low user interaction high throughput and low experimental variation For an introduction of DeCyder 2D see Chapter 8 For a detailed guide see DeCyder 2D Software User Manual To compare protein spot volumes across a range of experimental samples and gels two distinct steps are required e Intra gel co detection of sample and internal standard protein spots e Inter gel matching of internal standard samples across all gels within the experiment Both of these analysis steps can be performed with minimal user intervention by DeCyder 2D 2 4 1 Intra gel co detection Three scans will be made of each gel Cy2 Cy3 and Cy5 scans Scanned images of each sample and the internal standard are overlaid in DeCyder 2D The algorithms within the software co detect the spots present in each scan effectively identifying the position of each spot within the gel Fig 2 4 The spot boundaries that result are identical for each image in the gel This minimizes variation from detection and background subtraction with the added benefit that every protein in the sample is intrinsically linked to the corresponding protein spot in the internal standard sample Ettan DIGE System User Manual 18 1173 17 Edition AB DIGE concepts 2 Gel 1 3 scans Sample 2 Cy5 Cy2 Cy 3 Cy5 Sample 1 Cy3 Standard Cy2 Fig 2 4 Intra ge
48. d protein spots from stained gels are automatically picked by Ettan Spot Picker using a pick list created from the image analysis software and the gel plugs are transferred into microplates See Ettan Spot Picker User Manual 2 Digestion of the picked proteins The gel plugs are first trypsin digested in Ettan Digester and the resulting peptides extracted from the gel plugs See Ettan Digester User Manual 3 Spotting of samples onto MALDI targets The extracted peptides are mixed with matrix solution and spotted on MALDI targets using Ettan Spotter See Ettan Spotter User Manual Ettan DIGE System User Manual 18 1173 17 Edition AB Preparative workflow 9 Ettan Spot Handling Workstation automatically processes and transfers biomolecules from polyacrylamide gels to MS targets A computer running proprietary software controls the whole process For detailed instructions see Ettan Spot Handling Workstation User Manual 9 9 Mass spectrometry analysis MALDI targets are analyzed by MALDI ToF MS for protein identification Time of flight mass spectrometry is a technique for analyzing molecular weights based on the motion of ionized samples in an electrical field In MALDI ToF a matrix bound sample is bombarded with a pulsed laser beam to generate ions for subsequent detection Protein identification by mass spectrometry is usually performed on spot picked unlabeled protein visualized on the second dimension SDS PAGE gel with a post electrop
49. desshssassiusisdesnbosiocioosdecopdeventeectGessdsscqpcshceee CyDye DIGE Fluor minimal dyes GES CHI UIOMN essi aa noe en Ra 16 18 labeling reaction stock solution dilution CyDuye DIGE Fluor saturation dyes IG CNIMAGIECCHOM sestszasasssieserssatbeacesctecedecesicastelaelaatiastesuarteniettnesiiceds ANN BRN NRA D DeECYCer 2 D SORWAMNE gotsssgartstsatss senares raise iine eaii O Deep Purple POST STING 1o4300 orsstssbioss aiana EAA N a ANS eSATA NGA re DIA s DIGE File Naming Format Displacing solution s s E EDA Equilibration of focused Immobiline DryStrips Equilibration solution doms Equilibration solution 2 Ettan DALT Ettan DIGE Imager uu as Ettan IPGphor 3 Isoelectric Focusing System Experimental design inter gel matching internal standard sel6 a20 27 IEF preparativ workflow aise rehydration of Immobiline DryStrips running on Ettan IPGphor 3 instrument running preparations ae sample application protocol selection ee WOTLKFIOW a eceessssseeeeeees ImageMaster 2D Platinum software Ettan DIGE System User Manual 18 1173 17 Edition AB 109 Index 110 ImageQuant TL cropping trouble shooting Immobiline DryStrip Ja UT Lolo dfol a taeee ee RESER NERE n neer re errant CRP rehydration Reswelling Tray L Labeling CyDye DIGE Fluor minimal dyes CyDye DIGE Fluor saturation
50. dyes testing sample labeling workflow Post staining Preparative gel mn Preparative gel staining Preparative workflow preparing the labeled protein samples for the first dimension R Reference markers ates teat eas tee ten a eee shee teen ea lead orale stan eo Goud EN 83 RESUL WCCO pentan irin h EKREN ARN AEAEE EAER 13 S Sample preparation cell wash solution preparative workflow preparing the labeled protein samples for the first dimension protein quantitation is FEQUIFEMENTS TOM IYSIS BUTTER crr RAG ed 30 Scanning emission filters file output fluorescence acquisition mode fluorescence scan parameters gel alignment guide gel orientation image cropping monitoring scan progress pixel size PMT voltage press sample select scan area sensitivity SDS electrophoresis running buffer oo SDS Equilibration buffer stock solution 54 59 Second dimension SDS PAGE CASUING GESni aanne aaan aa a ia annA 52 Ettan DIGE System User Manual 18 1173 17 Edition AB Index loading of focused Immobiline DryStrips low fluorescence glass plates preparative workflow running buffer storage of gels post electrophoresis storage of gels prior to separation Standard cell lysis solution T TROUBIESHOOLIAGi r E E E EA Typhoon Variable Mode Imager cleaning instrument pl
51. e CyDye DIGE Fluor minimal dyes reducing the concentration of dye available for protein labeling 1 Take the CyDye DIGE Fluor minimal dye from the 20 C freezer spin briefly to ensure that the powder is at the bottom of the tube and leave to warm for 5 min at room temperature without opening This will prevent exposure of the dye to condensation which may cause hydrolysis 2 Take a small volume of DMF from its original container and dispense into a microfuge tube 3 From this tube remove the specified volume of the aliquoted DMF see specification sheet supplied with the CyDye DIGE Fluor dye and add to each new vial of dye Recommendations for e CyDye DIGE Fluor minimal dyes 25 ul DMF to 25 nmol of dye 1 mM e CyDye DIGE Fluor saturation dyes 50 ul DMF to 100 nmol of dye 2 mM 4 Replace the cap on the microfuge tube containing the dye and vortex vigorously for 30 seconds to dissolve the dye 5 Centrifuge the microfuge tube for 30 seconds at 12 000 x g in a benchtop microfuge CyDye DIGE Fluor saturation dye stock solution 2 mM can be used with no further dilutions Once reconstituted the saturation dye stock solution is stable for three months CyDye DIGE Fluor minimal dye stock solution 1 nmol ul is prepared but should be diluted before use Once reconstituted the minimal dye stock solution is stable for three months or until the expiry date on the container whichever is sooner Ettan DIGE System User Manual 18 117
52. e chapter 3 Compensation for system related result variation System variation cannot be overcome when using conventional 1 color 2D electrophoresis but by using Ettan DIGE System it is possible to minimize the gel to gel variation effects on results e In small experiments it is possible by multiplexing to run all samples on the same gel and thereby completely eliminate gel to gel variation Ettan DIGE System User Manual 18 1173 17 Edition AB 13 2 DIGE concepts 2 2 CyDye DIGE Fluor dyes 14 e In larger experiments a combination of multiplexing of samples and inclusion of an internal standard within each gel all system related result variation can be adjusted for Any protein spot can be compared and normalised to any other corresponding protein spot in the same or any other gel in which the same internal standard has been used Differentiation of inherent biological variation from induced biological change Biological variation cannot be removed from any 2D electrophoresis experiments However Ettan DIGE system allows the inherent biological variation to be effectively differentiated from induced biological changes using highly accurate measurement of protein abundance changes an appropriate experimental design and advanced statistical analysis The use of biological replicates in the experimental design ensures a true measurement of induced biological differences above the background of inherent biological variation Ettan DIGE
53. e container to create a dark environment and incubate for 1 hour at room temperature with gentle agitation Note The solution is light sensitive and should be kept out of bright light Note Containers can be wrapped in foil or covered with black plastic It is not necessary to eliminate light completely only to ensure that bright light is significantly reduced Alternatively containers with lids that are a solid colored plastic may be used 7 Pour off the stain and replace with 7 5 v v acetic acid Cover the container to create a dark environment and incubate with gentle agitation for at least 15 minutes 8 Repeat the acetic acid step once The gel can be imaged at this stage Note If speed is more important than background levels the gel can be imaged after one acetic acid step Further washes in acetic acid can be performed to reduce the background still further if necessary After imaging the gels can be stored in the dark in 7 5 v v acetic acid at 2 8 C for several weeks This allows for further imaging at a later date if required 9 6 Gel scanning We recommend using Typhoon Variable Mode Imager or Ettan DIGE Imager Please refer to Chapter 7 for more details 1 Place the gel glass side down onto a clean dust free platen surface 2 Image the gel with the appropriate filter set and exposure times It is recommended that the image resolution for the analytical and preparative gels are set at the same level and are
54. e purpose of the experiment the desired pH range of the Immobiline DryStrips and sample protein amount and concentration see Table 5 1 For other types of sample application protocols see 2D Electrophoresis Principles and Methods handbook Ettan DIGE System User Manual 18 1173 17 Edition AB First dimension isoelectric focusing IEF 5 Table 5 1 Sample application protocol selection criteria Experimental conditions or sample type Recommended protocol Samples sensitive for e g proteolysis protocol minimizes the time the sample is exposed to room temperature Cup loading protocol Samples with volumes up to 150 ul or with a protein amount under 150 ug Large sample loads increase the risk for protein precipitation at the point of application Cup loading protocol Samples with volumes larger than 100 ul e g diluted samples Rehydration loading protocol Experiments using basic Immobilline DryStrips pH 6 9 pH 6 11 pH 7 11 NL Cup loading protocol or Paper bridge loading protocol see 2D Electrophoresis Principles and Methods handbook for protocol Samples for preparative electrophoresis and for large sample loads above 150 ug protein Rehydration loading protocol or Paper bridge loading protocol see 2D Electrophoresis Principles and Methods handbook for protocol 5 3 Rehydration and sample application There are two protocols available for rehydration of Immobiline DryStri
55. e quantify reagent and re run 1D Test against recommended samples Fig A 3 Decision tree for troubleshooting labeling using 1D gels Ettan DIGE System User Manual 18 1173 17 Edition AB 95 A Testing cell lysates for successful labeling A 2 Recipes A 2 Recipes 2x Gel loading buffer Reagent Quantity Final concentration Tris 1 M pH 6 8 12 ml 120 mM Glycerol 87 v v 23 ml 20 v v SDS MW 288 38 4g 4 w v DTT MW 154 2 3g 200 mM Bromophenol Blue A few grains trace Make up to 100 ml with distilled water 12 5 1D SDS PAGE gel composition Reagent Quantity for 100 ml of a 12 5 gel Acrylamide Bis 40 w v 32 0 ml Tris 1 5M pH 8 8 25 0 ml 10 w v SDS 1 0 ml 10 w v APS 1 0 ml undiluted TEMED 40 ul add immediately prior to use Make up to 100 ml with distilled water 1x SDS electrophoresis running buffer Reagent Quantity Final concentration Tris MW 121 14 60 5 g 25mM Glycine MW 75 07 288 g 192 mM SDS MW 288 38 20g 0 1 w v Make up to 20 I with distilled water Store at room temperature for up to 3 months 96 Ettan DIGE System User Manual 18 1173 17 Edition AB Appendix B Labeling of cell surface proteins B Labeling of cell surface proteins B 1 Selective labeling of cell surface proteins Cell surface proteins can be difficult to detect in a second dimension SDS PAGE gel without fractionation or some other
56. easily view compare analyze and present your results The powerful suite of features that normally apply to 2D gels in ImageMaster can now be used in conjunction with DIGE gels by using the lmageMaster 2D Platinum 6 0 DIGE module of the software This section briefly outlines the features and capabilities of the software For a detailed guide please refer to the ImageMaster 2D Platinum User Manual which also includes a series of tutorials designed to provide new users with the means to gain a rapid understanding of the software s capabilities An online help is also integrated with the software 8 2 2 Image analysis workflow A typical image analysis using ImageMaster 2D Platinum software for DIGE gels would consist of the steps below For detailed information see the ImageMaster 2D Platinum User Manual 1 Acquiring data Gel images must first be digitized using an image capture device This will generally be done with a separate software Open gels from TWAIN compatible scanners with ImageMaster 2 Setting up a workspace Set up a workspace to open and work on gel images The workspace allows to organize gels into projects to define match sets and classes and to keep accompanuing data such as reports and image documents in project related folders Preferred ImageMaster settings are also saved in the workspace file 3 Visualizing gels This step is to handle the gel files open save print close manipulate the gel images select
57. eieindirtsnaintettintiacnteitaisanitadnieeeamaaes 29 4 2 WOKO W remi an a aai a 30 4 3 Sample preparationen aan nn 30 4 3 1 Solution recommendatioNS ssiissisinririnsiisienisnisisis 30 4 3 2 Protocol 4a FACILIS TNE aassssscscadtccsscivassassssesosiososcnsevzsonsasaaasenstaraessobranvoas aussoiesdcaceasvanvass 32 4 5 Protein concentration determination s mmemmmsssmmm 33 4 6 Internal standard preparation csssssssessssssssssssssssusssesssssscsscssssssssseesesees 33 Ettan DIGE System User Manual 18 1173 17 Edition AB V Contents vi yT Lapeliai ann Seen vaser NR EN ess ris 4 7 1 Preparation of CyDye DIGE Fluor dyes for labeling 4 7 2 Preparation of working dye solution esses a 4 7 3 CyDye DIGE Fluor Minimal dye labeling 4 7 4 CyDye DIGE Fluor saturation dye labeliNG z 2 lt smmemsmsmmmusmsmsmsms 4 8 First dimension sample preparation 4 9 RECIPES obest cect A tater ee rete sleet eo First dimension isoelectric focusing IEF 5 1 Ettan IPGphor 3 Isoelectric FOCUSING System n 41 5 1 1 General precautions for good results 5 2 Wor ROW rinner ere sinar serna n Hr 5 2 1 Sample application protocol selection s s sssssumsmmmmummmsnn 42 5 3 Rehydration and sample application 5 3 1 Rehydration of Immobiline DryStrips 5 3 2 Preparations for first dimension run including Cup loading 45 5 4 First dimension isoelectric focusing
58. ents tested for compatibility with Ettan DIGE system C Phosphatase inhibitors Phosphatase inhibitor cocktail 1 Sigma Compatible at manufacturer s recommended concentrations Phosphatase inhibitor cocktail 2 Sigma Compatible at manufacturer s recommended concentrations Sample preparation kits 2D Clean up kit Compatible at manufac concentrations urer s recommended SDS PAGE Clean up kit Compatible at manufac concentrations urer s recommended 2D Quant kit Compatible at manufac concentrations urer s recommended Sample grinding kit Compatible at manufac concentrations urer s recommended Ettan DIGE System User Manual 18 1173 17 Edition AB 101 C Reagents tested for compatibility with Ettan DIGE system C 1 List of reagents 102 Ettan DIGE System User Manual 18 1173 17 Edition AB Appendix D Ettan DIGE System User Manual 18 1173 17 Edition AB Trouble shooting guide D Trouble shooting guide The aim of this Appendix is to provide a help guide for problems that might be encountered when running experiments For general 2D troubleshooting problems please refer to 2D Electrophoresis Principles and Methods handbook D 1 Sample preparation and labeling Problem Cause Remedy Low protein Lysis procedure Ensure cell culture densities were yields from optimal for cell lysis using sonication the cell lysate Ensure cell pellet was not
59. essseeeesseeessseees 2 1 1 2D analysis result variation a sme membbmbbmsmsmeswsessumsussussm 2 1 2 Improvement of results by use of Ettan DIGE system 2 2 CyDye DIGE Fluor dyes on sssssssssesessssssccssssssssssssesessccsecessssees 2 21 CyDye DIGE Fluor minimal CYGSomssimmsmensnssnsnnesnesnnsnnssersnrsnnennrnnr enn nnn nn nn 2 2 2 CyDye DIGE Fluor saturation dyes sm 2 3 The internal standard 2 3 1 Advantages of using an internal Stan COOL 2 3 2 Examples of benefits using an internal StCANGOC hu 2 4 Co detection and matching using DeCyder 2D 2 4 1 Intra gel co detection sm 2 4 2 Inter gel matching 25 PFOtelnabBUnGONCE sssssnrs eesti innannndsdst annenin a 2 6 Statistical tests of protein abundance in DeCyder 2D 24 3 Experimental design SL INtrOMUCHON sssini ncn dian ania 25 3 2 Designing 2D DIGE experiMents mu essswmmssswsssswssssssssssmsssms 25 3 2 1 Internal standard sample on each JO m e msmmsmmumemsssmmssms 25 3 2 2 Biological replicates 3 2 3 Randomization Of samples escessssssssessssesessssssssssssssssssssassssssssssscesesseeeeeeseeseees 26 3 2 4 No gel replicates of the same sample is needed a 26 3 3 Examples of experimental design 3 3 1 CyDye DIGE Fluor minimal AYES cccsssssssssssssssssssssssssssssssssssssscsessssseeeesesseees 3 3 2 CyDye DIGE Fluor saturation dyes s mmeememummmmmeumsmsmmmmsssmsms 27 4 Sample preparation and labeling 41 MEFOCUGUOMN isinst
60. for 15 minutes The samples are now ready for the first dimension isoelectric focusing step Note After adding 2x sample buffer and incubating on ice it is recommended that the sample is run immediately on Immobiline DryStrips Proceed to Chapter 5 First dimension isoelectric focusing IEF 4 9 Recipes Cell wash solution Reagent Quantity Final concentration Tris 100 mM pH 8 0 5 0 ml 10 mM Magnesium acetate 1 M 0 25 ml 5mM Make up to 50 ml with distilled water Store at 4 C Stable for 1 month 38 Ettan DIGE System User Manual 18 1173 17 Edition AB Sample preparation and labeling 4 Cell lysis solution 1 Decide whether to use cell lysis solution 1 or 2 The cell lysis solution option 1 is very similar to option 2 except that thiourea is added which has been shown to solubilize many more proteins especially membrane proteins 2 Adjust the whole solution to pH 8 5 using dilute HCI Confirm the pH of your cell lysis solution by spotting 5 ul on a pH indicator strip 3 Make the volume of the cell lysis solution up to 100 ml 4 The cell lysis solution can now be aliquoted and stored at 20 C Cell lysis solution option 1 contains thiourea Reagent Quantity Final concentration Urea MW 60 06 42 0g 7M Thiourea MW 76 12 15 22 g 2M Tris 1M not pH d 3 0 ml 30 mM CHAPS MW 614 89 4g 4 w v Make up to 100 ml with distilled water Small aliquots can be stored at 20
61. ge to collect the solution at the bottom of the tube Leave on ice for 30 min in the dark 5 Add 1 ul of 10 mM lysine to stop the reaction Mix and spin briefly in a microcentrifuge Leave for 10 min on ice in the dark Labeling is now finished The labeled samples can be processed immediately or stored for up to 3 months at 70 C in the dark 36 Ettan DIGE System User Manual 18 1173 17 Edition AB Sample preparation and labeling 4 4 7 4 CyDye DIGE Fluor saturation dye labeling The dye labeling reaction is designed to be simple and should take about 2 h to perform Recommended conditions e tis very important that the cell lysate protein sample has pH 8 0 in order to achieve an efficient labeling see section 4 4 e TCEP dye concentration ratio should be kept at a 1 2 ratio to ensure efficient labeling e Protein concentration of 0 55 10 mg ml in the cell lysate e For samples containing proteins of interest with high cysteine content more TCEP reduces disulphide bonds and more dye for labeling thiol groups are required Protocol 1 Adda volume of protein sample equivalent to 5 ug to a microfuge tube 2 Make up to 9 ul with cell lysis solution 3 Add1ul2mMTCEP 4 Mix vigorously by pipetting and spin Note Since cell lysates are viscous it is important to mix samples thoroughly in this and all following mixing steps to avoid non uniform labeling 5 Incubate at 37 C for 1h in the dark 6 Add 2 ul 2 mM C
62. h CyDye DIGE Fluor you Ev ww Samples Pool portion 1s Y Y Y Labeling with CyDye DIGE Fluor y minimal dyes y n Internal standard Fig 4 1 Sample preparation and labeling with CyDye DIGE Fluor minimal dyes Recommended protocols for preparation of a cell lysate and labeling are presented in sections 4 3 to 4 8 and recipes for recommended buffers and solutions are given in section 4 9 Note Some standard methods for preparation of protein samples for conventional 2D electrophoresis may not be compatible with Ettan DIGE system Ettan DIGE System User Manual 18 1173 17 Edition AB 29 4 Sample preparation and labeling 4 2 Workflow 30 4 2 Workflow Workflow for sample preparation and labeling 1 Sample preparation Prepare cell lysates compatible with CyDye DIGE Fluor minimal dye or CyDye DIGE Fluor saturation dye labeling 2 Adjust the pH of the cell lysates 3 Determine cell lysates protein concentration 4 Prepare an internal standard 5 Label protein samples using CyDye DIGE Fluor minimal dyes or CyDye DIGE Fluor saturation dyes 6 Prepare labeled protein samples for first dimension isoelectric focusing 4 3 Sample preparation The sample preparation protocol in this section is designed to use cell cultures as starting material For sample preparation protocols for other starting material see www ettandige com 4 3 1 Solution recommendations Cell wash solution It is recommended to use the
63. horesis stain such as Deep Purple Some applications may require direct spot picking from a second dimension SDS PAGE gel containing protein labeled with CyDye DIGE Fluor minimal dye The nature of the minimal labeling approach results in the majority of the protein and peptide population remaining unlabeled The results can be imported into DeCyder 2D software modules BVA and EDA for further analysis in the EDA module 9 10 Recipes Recipes for post staining of gels Bind Silane working solution Reagent Quantity Ethanol 16 ml Glacial acetic acid 400 ul Bind Silane 20 ul Double distilled H2O 3 6 ml Ettan DIGE System User Manual 18 1173 17 Edition AB 87 9 Preparative workflow 9 10 Recipes Fixation solution Reagent Quantity Final concentration Ethanol 100 ml 10 v v Acetic acid 75 ml 7 5 v v Make up to 1 000 ml with distilled water 20 fold excess of the gel volume should be used Wash solution small usually free floating gels Reagent Quantity Final concentration Na CO3 2129 200 mM Dissolve NazCO3 in 750 ml water Make up to 1 000 ml with distilled water The pH of the solution should be at least 11 and should be verified 20 fold excess of the gel volume should be used This solution can be stored for up to 2 weeks Wash solution large usually backed gels Reagent Quantity Final concentration NaHCO 2 94g 35 mM Na CO3 3189 300 mM Dissolve NaHC
64. in the DeCyder database making them accessible for other modules The import must be performed before analyses can be performed in DeCyder 2D software Ettan DIGE System User Manual 18 1173 17 Edition AB 75 8 Image analysis 8 1 DeCyder 2D software 76 Batch Processor Fully automated image detection and matching of multiple gels without user intervention Proteins of interest can be automatically filtered and picking list generated DIA Differential In gel Analysis Protein spot detection and quantitation on a set of images from the same gel Features include background subtraction in gel normalization and gel artefact removal Images must be processed in the DIA interface prior to data analysis in BVA BVA Biological Variation Analysis Matching of multiple images from different gels to provide statistical data on differential protein abundance levels between multiple groups XML Toolbox Extraction of user specific data from XML files generated in either the Batch DIA or BVA modules This data can be saved in either text or html format enabling users to access data from DeCyder 2D workspaces in other applications EDA Extended Data Analysis Multivariate analysis of data from several BVA workspaces EDA is an add on module for the DeCyder 2D software and can handle up to 1000 spot maps The raw data gel images are linked to EDA and can be opened for display via the BVA module In addition to the univariate analyses that
65. ing as to whether this change is above the biological variation Statistical tests can then be applied to the data in DeCyder 2D for example Student s T test and ANOVA The statistical tests compare the average ratio and variation within each group to the average ratio and variation in the other groups to see if any change between the groups is significant If using the Extended Data Analysis EDA module of DeCyder 2D additional multivariate statistical analyses such as Principal Component Analysis PCA Pattern Analysis and Discriminant Analysis can be performed Ettan DIGE System User Manual 18 1173 17 Edition AB Experimental design 3 3 Experimental design 3 1 Introduction Prior to commencing practical work experimental design needs to be carefully considered This chapter addresses experimental design unique to 2D DIGE analysis for optimal data analysis using DeCyder 2D software 3 2 Designing 2D DIGE experiments When designing 2D DIGE experiments the following recommendations should be considered 1 Inclusion of an internal standard sample on each gel 2 The requirement for biological replicates such as multiple cultures tissue etc 3 Randomization of samples to produce unbiased results thus conforming with best experimental practice 4 No gel replicates of the same sample is needed 3 2 1 Internal standard sample on each gel It is recommended that an internal standard is run on all gels within an experiment as it
66. internal standard shows that gel to gel variation has resulted in an increased spot volume in gel B compared to gel A This means that instead of an increasing trend in spot volume from samples 1 to 4 the relative volume of the protein spot in sample 3 is reduced in comparison to samples 1 2 and 4 where the spot volume ratios are identical Standard Cy2 Sample 1 Cy3 Sample 2 Cy5 Sample 3 Cy3 Sample 4 Cy5 With internal standard 1 2 3 4 Gel to gel variation or induced biological change Gel Cy2 Standard Cy3 Cy5 A Pool samples 1 4 Sample 1 untreated Sample 2 treated B Pool samples 1 4 Sample 3 treated Sample 4 untreated Fig 2 3 Example to illustrate the benefits of an internal standard in correctly identifying differences between samples 1 2 3 and 4 The right panel shows the different results achieved of the volumes of the protein spots without and with an internal standard Ettan DIGE System User Manual 18 1173 17 Edition AB 19 2 DIGE concepts 2 4 Co detection and matching using DeCyder 2D 20 2 4 Co detection and matching using DeCyder 2D To capitalize on the ability to multiplex and use an internal standard DeCyder 2D software has been specifically designed for the Ettan DIGE system DeCyder 2D contains proprietary algorithms that perform co detection of differently labeled samples within the same gel DeCyder 2D also permits automated detection background subtra
67. is then possible to minimize effects of system related result variation see section 2 3 The analysis of results then allows the inherent biological variation to be effectively differentiated from induced biological changes using appropriate experimental design and statistical analysis Quantitative comparisons of protein between samples are made on the relative change of each protein spot to its own in gel internal standard This removes gel to gel system variation a common problem with conventional one sample per gel 2D studies It also enables accurate statistical quantification of induced biological change between samples Ettan DIGE System is the only protein difference analysis technique that utilises this approach 3 2 2 Biological replicates It is strongly advised that biological replicates are included in every group By increasing the number of biological replicates it is possible to get an accurate measurement of the change due to a treatment or disease that is significant above a baseline of inherent biological variation Gel replicates of the same biological sample will not deliver this information Without biological replicates results may not be biologically relevant and it is often only possible to conclude that differences in results are above system variation Ettan DIGE System User Manual 18 1173 17 Edition AB 25 3 Experimental design 3 3 Examples of experimental design 26 3 2 3 Randomization of samples Random
68. ization of samples across gels removes any bias from the experiments such as experimental conditions sample handling and labeling ensuring that results from 2D DIGE experiments are accepted by peers Even if the system related result variation is low using Ettan DIGE System it is good laboratory practice to distribute individual experimental samples evenly between different CyDye DIGE Fluor dyes and different gels to avoid for example systematic errors See Section 3 3 for examples 3 2 4 No gel replicates of the same sample is needed As the result variation using Ettan DIGE System is so low due to the internal standard and method of analysis any system variation will by far be outweighed by the inherent biological variation However gel replicates can be included if desired 3 3 Examples of experimental design In order to maximize the value of CyDye DIGE Fluor dyes and DeCyder 2D software it is important to carefully consider the experimental testing regime Two examples using CyDye DIGE Fluor minimal dyes and CyDye DIGE Fluor saturation dyes sections 3 3 1 and 3 3 2 respectively are presented below to illustrate examples of experimental design 3 3 1 CyDye DIGE Fluor minimal dyes Comparison of protein abundance between three differently treated samples A C each with four biological replicates using e CyDye DIGE Fluor Cy2 Cy3 and Cy 5 minimal dyes e Internal standard labeled with CyDye DIGE Fluor Cy2 minimal dye e A design
69. king concentration 0 4 mM Reducing step TCEP prior to labeling o reduction step required Protein labeling Labeling reaction at 37 C Labeling reaction quenched with 2x sample buffer Labeling optimization by titrating TCEP and dye analysis on 1D SDS PAGE gel Labeled proteins stable 1 month at 70 C Labeling reaction on ice Labeling reaction quenched with 10 mM lysine Labeling optimization by comparing labeled samples analysis on 1D SDS PAGE gel Labeled proteins as stable as unlabeled proteins at 70 C Protein separation and analysis o iodoacetamide equilibration step prior to second dimension electrophoresis A Cy 3 labeled sample is used on preparative gel for spot picking odoacetamide equilibration step required prior to second dimension electrophoresis An unlabeled sample is used on preparative gel for spot picking Post staining is required for matching to analytical gels 2 2 1 Chemical description CyDye DIGE Fluor minimal dyes CyDye DIGE Fluor minimal dyes are three spectrally resolvable dyes Cy 2 Cy3 and Cy5 matched for mass and charge Each CyDye DIGE Fluor minimal dye when coupled to a protein will add 450 Da to the mass of the protein This mass shift does not effect the pattern visible on a second dimension SDS PAGE gel A protein labeled with any of the CyDye DIGE Fluor minimal dyes will migrate to the same position on the second dimension SD
70. l co detection All samples are co detected with the internal standard 2 4 2 Inter gel matching Experimental design ensures that each gel contains the same internal standard This enables inter gel comparisons of spot abundance Before this can be done it is important to ensure that the same protein spots are compared between gels DeCyder 2D achieves this using the internal standard to match the position of each protein across all gels within the experiment The internal standard image with the most detected spots is assigned as the Master Following co detection each image has a spot map species The spot map species for the internal standard assigned as the Master is used as a template to which all remaining spot map species for the other internal standards intrinsically linked to their co detected sample images are matched Fig 2 5 Ettan DIGE System User Manual 18 1173 17 Edition AB 21 2 DIGE concepts 2 5 Protein abundance 22 Matching gel 1 master to gel 2 standard Matching gel 1 master to gel 3 standard Fig 2 5 Inter gel matching only the internal standards need to be matched These are derived from the same sample which aids matching 2 5 Protein abundance Once the protein spots have been matched the ratio of protein abundance between samples can be determined The use of an identical internal standard within all the experimental gels enables a comparison of protein abundance between samples on diffe
71. lost after centrifugation Ensure sonication was carried out for long enough Insufficient protein in Remake protein lysate or concentrate sample protein con sample by precipitation with Ettan 2D centration lt 1 mg ml Clean Up Kit code no 80 6484 51 Horizontal Low pH prior to Check that pH is within range 8 9 streaking labeling immediately prior to labeling Unexpected Contaminantproteins Check that gloves are used throughout protein have been introduced the procedure spots into the sample prior present in to the labeling the gel reaction Protein Proteins not Use combination of chaotrope in lysis spots denatured or buffer such as 7 M urea 2 M thiourea detected solubilized sufficiently more strongly with one dye 103 D Trouble shooting guide D 1 Sample preparation and labeling Problem Cause Remedy Weak fluorescent Insufficient sample buffering Use 30 mM Tris to give sufficient buffering capacity signal on 2D gel image Thiol agents present in the sample competing for dye Check if concentration of DTT is gt 2 mg ml in protein sample preparation method Dilute protein lysate with DTT free lysis buffer Clean sample by precipitation with Ettan 2D Clean Up Kit or increase the amount of dye in the labeling reaction Primary amines such as Pharmalytes or ampholytes are present in sample during labeling competing for CyDye DIGE Fluor minimal dye Dilute protein lysate with amine free
72. lts mmsesmumsmsmemmm 108 Index Ettan DIGE System User Manual 18 1173 17 Edition AB vii Contents vill Ettan DIGE System User Manual 18 1173 17 Edition AB 1 Ettan DIGE System User Manual 18 1173 17 Edition AB Introduction 1 Introduction 1 1 Ettan DIGE System overview Ettan DIGE system is based on the technique of two dimensional difference gel electrophoresis 2D DIGE It is a powerful tool for separating complex mixtures of proteins by charge and size and for scanning and analyzing the resulting second dimension SDS PAGE gel images for protein differences Combining novel proprietary technologies in fluorescence sample multiplexing and image analysis Ettan DIGE System is a fully integrated system offering significant benefits over classical second dimension SDS PAGE The system comprises CyDye DIGE Fluor minimal dyes labeling kit for scarce samples Typhoon Variable Mode Imager and DeCyder 2D software Alternatively Ettan DIGE Imager can be used instead of Typhoon Variable Mode Imager and ImageMaster 2D Platinum software can be used instead of DeCyder 2D software The use of CyDye DIGE Fluor minimal dyes enables multiplexing of up to three separate protein mixtures on the same second dimension SDS PAGE gel The multiplexing capability of the 2D DIGE methodology enables the incorporation of the same internal standard on every gel and thereby eliminates gel to gel variation Ensuring that each
73. lutions are similar Follow protocols in sections 4 7 1 and 4 7 2 for both types of dyes but use the appropriate section of 4 7 3 and 4 7 4 Note Itis recommended that all new cell lysates or samples containing chemical components that has not been approved for DIGE use are checked for successful labeling see Appendix A 4 7 1 Preparation of CyDye DIGE Fluor dyes for labeling The reconstitution and storage of CyDye DIGE Fluor minimal and saturation dyes is important to the success of sample labeling If reagents such as dimethylformamide DMF are of a low quality or the CyDye DIGE Fluor minimal dyes are incorrectly stored protein labeling will not be efficient Ettan DIGE System User Manual 18 1173 17 Edition AB 33 4 Sample preparation and labeling 4 7 Labeling 34 Reconstituting CyDye DIGE Fluor dye in dimethylformamide CyDye DIGE Fluor minimal and saturation dyes are supplied as a solid powder and are reconstituted in dimethylformamide DMF giving a concentration of 1 nmol l After reconstitution in DMF the dye will give a deep color Cy2 yellow Cy3 red and Cy5 blue Note Itis recommended that a new bottle of DMF is opened every 3 months The DMF must be high quality anhydrous Specification lt 0 005 H20 99 8 pure Sigma Aldrich 22 705 6 and every effort should be taken to ensure it is not contaminated with water Note DMF once opened will start to degrade generating amine compounds which will react with th
74. mage acquisition 7 3 Position the main bar of the Gel Alignment Guide onto the platen grippers L i front location bar 4 Using the grippers position the dried glass plate assembly with one edge on the spacer and against the front location bar and gently lower the assembled gel onto the platen 5 Position the gel by using the Gel orientation guide The physical gel orientation should be noted by the user the gel orientation option in the software determines the file output orientation Basic Acidic I IPG strip High Mw Gel Low Mw Tip Using Ettan DALT trays it is recommended that Ettan DALT gels are scanned with the short glass plate facing down to the platen Ettan DIGE System User Manual 18 1173 17 Edition AB 63 7 Image acquisition 7 5 Scan parameters and scanning 6 Close the instrument lid 7 5 Scan parameters and scanning Start the Typhoon Scanner Control software The Typhoon Scanner Control Multiple Sample scan window is opened Fig 7 2 File Templates Tray Help Scanner Information m Setup Modet Typhoon9410 Address 192 168 1 1 Serial Number 94409 User Name bb Instrument State Recon Sak Ready Initialize Scanner EL SES Template Min Cy2 3 5 Ti ma DIGE Ettan DALT 2 a i
75. mages of 2D DIGE applications Fig 7 3 In particular it can create high quality images of 2D DIGE gels By combining very high resolution with precise motion control the Ettan DIGE Imager produces accurate multi channel images of Cy2 Cy3 and Cy5 labeled gels The system has also been designed to image a wide range of other fluorescent gel and membrane applications Fig 7 3 Ettan DIGE Imager The imager is controlled using Ettan DIGE Imager software and can be set up for a variety of gel and membrane formats Data produced by Ettan DIGE Imager is directly compatible with ImageQuant TL ImageMaster 2D Platinum and DeCyder 2D For information about how to use the imager refer to Ettan DIGE Imager User Manual Ettan DIGE System User Manual 18 1173 17 Edition AB 73 7 Image acquisition 7 7 Ettan DIGE Imager 74 Ettan DIGE System User Manual 18 1173 17 Edition AB 8 Image analysis 8 Image analysis oh amp Two different softwares have been specially designed for image analysis in Ettan DIGE System DeCyder 2D and ImageMaster 2D Platinum software DeCyder 2D is recommended in complex experiments when more than two groups or conditions are used in an experiment ImageMaster 2D Platinum software is suitable for basic experiments containing control vs treated or healthy vs non healthy conditions These dedicated 2D software products use the internal standard to minimize gel to gel result variation A detection of less than 1
76. mmended Application of sample via cup loading lt 50 mM recommended Ettan DIGE System User Manual 18 1173 17 Edition AB 99 C Reagents tested for compatibility with Ettan DIGE system C 1 List of reagents Buffers Tris Recommend 10 40 mM pH 8 0 9 0 pH is very important pH 8 5 is optimal HEPES Can cause focusing problems therefore not recommended Bicarbonate 5 mM pH 8 5 is acceptable CHES 5 mM pH 9 9 5 is acceptable PBS 150 mM phosphate buffered saline Tris sucrose 250 mM sucrose 10 mM Tris Protease Inhibitors For all protease inhibitors Mix compatible proteases at recommended concentrations 4 2 aminoethyl benzenesulphonyl fluoride AEBSF Pefabloc Causes charge trains unless protector reagent is used Complete protease inhibitor cocktail This product contains AEBSF so the same restrictions as above apply Aprotinin Compatible at recommended concentrations 4 amidino pheny methane sulphonuyl fluoride APMSF Compatible at manufacturer s recommended concentrations EDTA Compatible between 0 5 10 mM Phenylmethylsulphonyl fluoride PMSF Compatible at manufacturer s recommended concentrations Pepstatin A Compatible at manufacturer s recommended concentrations Protease inhibitor mix Compatible at recommended concentrations 100 Ettan DIGE System User Manual 18 1173 17 Edition AB Reag
77. move zoom stack align possibly transform the gels rotate crop scale and view the signal intensity adjust contrast profile 3D view Ettan DIGE System User Manual 18 1173 17 Edition AB 77 8 Image analysis 8 2 ImageMaster 2D Platinum software 10 78 Detecting and quantifying spots Perform automatic spot detection It is also possible to select display and edit spots as well as view spot properties and quantification values Annotating spots and pixels Individual pixels and spots in a gel image may be labeled with annotations These annotations can be used in functionalities such as calibration alignment and matching or be utilized to mark spots with their particular characteristics Create use select and display labels categories and annotations It is also possible to create links to external databases or data sources of any format text file html etc Matching gels After spots were detected and match sets defined gel images can be matched Analyzing data Data analysis and classifications tools to study the variations in protein expression among gels or classes of gels can be performed The data analysis step may be carried out at two different levels The intra class statistics tools include scatter plots descriptive statistics and factor analysis For inter class analyses the so called overlapping measures and various statistical tests can be used Heuristic clustering can help finding classes Integr
78. nsion electrophoresis Tei HAA 4 Scan the gels a EL Fm feel See xe Gel images Bd ed PY 5 Perform image analysis DeCyder 2D Software A AAA lt A lt lt E Proteins of interest Fig 1 1 Analytical workflow in Ettan DIGE System Labeling is performed using CyDye DIGE Fluor minimal dyes First dimension and second dimension SDS PAGE are performed using Ettan IPGphor 3 and Ettan DALTtwelve or Ettan DALTsix Scanning is performed using Typhoon Variable Mode Imager and image analysis is performed in DeCyder 2D software 10 Ettan DIGE System User Manual 18 1173 17 Edition AB Introduction 1 1 2 2 Preparative workflow The preparative workflow is used to perform spot picking and protein identification of the proteins of interest found in the analytical workflow To perform this a pick list containing the coordinates for the proteins of interest to pick is created and a pick gel preparative gel is prepared See Chapter 9 for detailed information about the different steps in the preparative workflow 1 3 Ettan DIGE System User Manual The scope of this user manual is to explain the central concept of the DIGE technology to give an understanding of the entire experimental workflow and an overview of the Ettan DIGE related products available Chapter 2 and 3 give a background to the DIGE system and to experimental design chapters 4 to 8 cover the DIGE system analytical workflow and chapter 9 contains
79. ntains DeStreak reagent and optimized concentrations of Urea Thiourea and CHAPS Note If IPGphor standard StripHolder or Immobiline DryStrips 7 11NL and 3 11NL are used use 0 5 IPGphor buffer instead of 2 IPGphor buffer 2x Sample buffer for CyDye DIGE Fluor minimal dyes Reagent Quantity Final concentration 2x Sample buffer 2 5 ml rehydration solution stock 1 or 2 IPG Buffer pH 3 10 50 ul 2 v v DTT MW 154 2 50 mg 2 w v 20mg ml 130 mM Do not store prepare fresh before use Use stock option 1 or 2 depending on the rehydration buffer required Note fusing DeStreak Rehydration solution the buffer may contain up to 10 mM DTT 2x Sample buffer for CyDye DIGE Fluor saturation dyes Reagent Quantity Final concentration 2x Sample buffer 2 5 ml rehydration solution stock IPG Buffer pH 3 10 25 ul 1 v v DTT MW 154 2 5 mg 0 2 w v 2 mg ml 13 MM Do not store prepare fresh before use Use stock option 1 or 2 depending on the rehydration buffer required Ettan DIGE System User Manual 18 1173 17 Edition AB 49 5 First dimension isoelectric focusing IEF 5 5 Recipes 50 Ettan DIGE System User Manual 18 1173 17 Edition AB Second dimension SDS PAGE 6 6 Second dimension SDS PAGE 6 1 Ettan DALT electrophoresis system Ettan DALT electrophoresis systems Ettan DALTsix and Ettan DALTtwelve are recommended for second dimension separation using sodium dodecyl sulfa
80. o cool slightly and slowly pipette the molten agarose solution along the upper surface of the gel up to the top of the glass plate Take care not to introduce bubbles Do not allow the agarose to solidify 5 Once the agarose solution has completely set the gel should be run in the second dimension as soon as practically possible Ettan DIGE System User Manual 18 1173 17 Edition AB 55 6 Second dimension SDS PAGE 6 6 Second dimension SDS PAGE 6 6 Second dimension SDS PAGE 1 Fill the lower buffer tank with SDS electrophoresis running buffer for Ettan DALT Turn on the control unit switch on the pump and set the temperature to 20 C Note To achieve good results it is important to use the recommended electrophoresis running buffer with 0 2 w v SDS 2 When the running buffer has reached the desired temperature insert the loaded gel cassettes with the Immobiline DryStrips in place Load the unit from back to front When all 12 slots are filled the buffer level should be slightly below the level of the buffer seal gaskets Note Blank cassette must be inserted into any unoccupied slots Alternate gel cassettes with blank cassette inserts to facilitate handling Tip Gel cassettes and blank cassette inserts slide much more easily into the unit if they are wet Use SDS electrophoresis running buffer for Ettan DALT from a wash bottle to wet the cassettes and inserts as they are being loaded 3 Pour SDS electrophoresis running buffe
81. o ensure that the nonionic detergent is present in a concentration at least 8 times higher than the concentration of any ionic detergent to ensure complete removal of SDS from the proteins Streaking or smearing Sample rich in nucleic acids Add DNase and RNase or sonicate to hydrolyze nucleic acids Sample aggregation or precipi tation Focusing conditions not optimized Program a low initial voltage that increases gradually and or increase time at maximum voltage Extended focusing may result in electro endosmosis where water and protein movement can produce horizontal streaking Minimize water transport by employing a maximum pH range Immobiline DryStrips and apply electrode pads Ettan DIGE System User Manual 18 1173 17 Edition AB 107 D Trouble shooting guide D 4 Typhoon Variable Mode Imager results D 4 Typhoon Variable Mode Imager results For a complete guide to troubleshooting Typhoon Imager results please refer to Typhoon User Guide Problem Cause Remedy Protein spots do not show up on the gel image The wrong laser and emission filters have been selected for the CyDye DIGE Fluor minimal dye used Select correct laser and filter for each CyDye DIGE Fluor minimal dye The labeling reaction has not been performed correctly Reconstitute stock dye or make fresh working dye solution in fresh DMF Repeat the labeling The PMT voltage is too low Rescan with
82. ol presented is adapted to Escherichia coli cell culture Other wash solution might be more appropriate for different cell types Approximately 4x1 ot E coli cells will result in 5 10 mg of protein in a total volume of 1 ml of standard cell lysis solution il 2 10 Pellet the cells in a suitable centrifuge at 4 C Pour off all growth media taking care not to disturb the cell pellet Re suspend the cell pellet in 1 ml of standard cell wash solution in a microfuge tube Pellet the cells in a bench top microfuge at 12 000 x g for 4 min at 4 C Remove and discard the supernatant Repeat steps 3 and 4 at least three times Ensure all the cell wash solution has been removed Re suspend the washed cell pellet in 1 ml of standard cell lysis solution and leave on ice for 10 min Note Ifthe protein concentration is less than 5 mg ml after protein quantitation see section 4 5 re suspend cells in a smaller volume of lysis solution in subsequent experiments Alternatively precipitate proteins using Ettan 2D Clean Up Kit code no 80 6484 51 and re suspend in a smaller volume of cell lysis solution Keep the cells on ice and sonicate intermittently until the cells are lysed Centrifuge the cell lysate at 4 C for 10 min at 12 000 x gina microcentrifuge Transfer supernatant to a labeled tube This is the cell lysate Discard the pellet Remove contaminating substances with Ettan 2D Clean Up Kit This procedure improves
83. onto the glass plate and to prevent the gel from deforming during the staining imaging and picking processes Ettan DIGE System User Manual 18 1173 17 Edition AB 81 9 Preparative workflow 9 4 Second dimension SDS PAGE The following protocol for treatment of glass plates was optimized for PlusOne Bind Silane Note Use the shorter glass plate without reference markers 1 82 Thoroughly scrape off any residual bound gel with a plastic scraper and wash the plate in 1 Decon v v branded Contrad in the USA with a soft sponge to further remove the gel Leave the plate to soak in 1 Decon v v overnight On the following day wash the plate with a soft sponge Rinse the plate with water and leave the plate to soak in 1 HCl v v for LM Wash the plate in 1 Decon v v with a soft sponge then rinse with double distilled water Dry the plate using a lint free tissue or leave to air dry in a dust free environment If not to be used immediately please store in a dust free environment Prepare the Bind Silane working solution Pipette 2 4 ml depending on plate size of the above solution over the whole surface of the plate and wipe it over with a lint free tissue until it is dry Cover the plate with a lint free tissue to prevent dust contamination and leave on the bench for 1 5 h minimum one hour for excess Bind Silane to evaporate Note Ifthe Bind Silane is not left to dry sufficiently before the glass plates
84. or sheets Leave 1 2 mm space from the edge uncovered Too much pressure will cause gel distortions Ettan DIGE System User Manual 18 1173 17 Edition AB Second dimension SDS PAGE 6 8 Add appropriate volumes of fresh APS to the acrylamide gel stock solution when ready to pour the gels and mix thoroughly Once the APS is added polymerization begins 9 Pour the gel solution until it is about 1 2 cm below the final desired gel height Fill the balance chamber with 100 ml of the displacing solution The dense displacing solution flows down the connecting tube filling the V well and sloped trough at the bottom of the caster The remaining acrylamide solution is forced into the cassettes to the final gel height 10 Fill the balance chamber with 100 ml of the displacing solution 11 Spray do not pipette overlay solution on the edges of the cassettes with for example a plant sprayer so the edges are covered with a few mm thick liquid layer using a 0 1 w v spray overlay solution By doing this curved edges on the gels are prevented Tip Alternatively 30 isopropanol can be used to overlay gels 12 Allow the homogeneous gels to polymerize for at least 3 h before disassembling the caster Best polymerization is achieved by letting the gel polymerize overnight at room temperature 13 Once gels are completely polymerized cover the top of the gel with SDS electrophoresis running buffer for the Ettan DALT Freshly made gels are best
85. ows the user to Set common details for all gels in a single operation The Browse option in both cases allows the user to select existing folders or file name structures A number of gels can be set up for a single scan run and obtain unique filenames for each gel image 15 Click SCAN to start the scan Once the scan has started the preview window appears e For unlinked scans a single image channel appears for each scan programmed the images appearing one at a time e For linked scans two image channels appear simultaneously e Where more than one gel is scanned using the DIGE Ettan DALT tray settings a drop down numerical menu appears allowing the user to monitor each of the gels as the images are generated Ettan DIGE System User Manual 18 1173 17 Edition AB 69 7 Image acquisition 7 5 Scan parameters and scanning 70 16 Evaluate the scan progress Saturated data is displayed in red in the preview window If saturated data is seen the user can cancel the scan and re set the PMT voltage without having to complete the whole scan 17 Handle image file output The image files created are labeled as filename gel This uses a modified 16 bit TIF format An additional text file filename ds also exists and this links image file data together for image overlays in ImageQuant TL 7 5 1 Pre scanning to identify a suitable PMT voltage The PMT voltage can be set from 300 to 1 000 V although it is recommended that where possible wo
86. pH of the lysate can be increased to pH 8 5 by careful addition of dilute sodium hydroxide 50 mM or higher Ettan DIGE System User Manual 18 1173 17 Edition AB Sample preparation and labeling 4 4 5 Protein concentration determination The concentration of protein in all lysates should be determined using a suitable protein assay compatible with detergents and thiourea if these are present in the cell lysate Protein Determination Reagent USB code no 30098 or Ettan 2D Quant Kit code no 80 6483 56 are both suitable for this activity 4 6 Internal standard preparation The internal standard is created by pooling an aliquot of all biological samples in the experiment and labeling it with one of the CyDye DIGE Fluor dyes usually Cy2 when using CyDye DIGE Fluor minimal dyes and Cy3 when using CyDye DIGE Fluor saturation dyes The internal standard is then run on every single gel along with each individual sample This means that every protein from all samples will be represented in the internal standard which is present on all gels In this way every protein spot on all gels will have an internal standard Note Sufficient internal standard must be prepared to allow enough to be included on every gel in the experiment 4 7 Labeling The protocols for labeling cell lysate samples with CyDye DIGE Fluor minimal and saturation dyes are different However the preparation of dyes including reconstituting and making working dye so
87. ple is ready for 2D electrophoresis Proceed with the recommended protocols for 2D DIGE electrophoresis Ettan DIGE System User Manual 18 1173 17 Edition AB Appendix C Reagents tested for compatibility with Ettan DIGE system C Reagents tested for compatibility with Ettan DIGE system This section contains examples of reagents commonly used in 2D electrophoresis experiments which have been tested for their compatibility with labeling using CyDye DIGE Fluor dyes They all have the DIGE approved seal attached This is not a complete list of reagents if unlisted reagents or a combination of these reagents are required in the cell lysis buffer it is recommended that the labeling efficiency is checked following the instructions in Appendix A These examples are only intended as a guide C 1 List of reagents Reducing agents DL dithiothreitol DTT 2 mg ml slight reduction in labeling 5 mg ml 2xreduction in labeling 10 mg ml 10x reduction in labeling CyDye DIGE Fluor minimal dyes will react with thiols at high concentration Tris 2 carboxyethyl phosphine TCEP 0 5 to 1 mM slight reduction in labeling 2 mM significant reduction in labeling B mercaptoethanol Significantly reduces labeling Detergents Triton X 100 use at 1 17 reduction in labeling NP40 up to 1 No effect on labeling SDS up to 1 No effect on labeling Salts Application of sample during rehydration lt 10 mM reco
88. ps and sample application Cup loading protocol and Rehydration loading protocol The protocols are rather similar The main difference is that with Cup loading protocol the samples are added to the strips after rehydration and with Rehydration loading protocol the samples are added to the strips during rehydration Note Some steps in the protocol below contain two options one to be used with the Cup loading protocol and the other with the Rehydration loading protocol 5 3 1 Immobiline DryStrips must be rehydrated before use For rehydration an Immobiline DryStrips Reswelling Tray is used The Immobiline DryStrips Reswelling Tray has 12 independent reservoir slots that each hold a single Immobiline DryStrip Separate slots allow the rehydration of individual Immobiline DryStrips in the correct volume of solution Rehydration of Immobiline DryStrips Ettan DIGE System User Manual 18 1173 17 Edition AB 43 5 First dimension isoelectric focusing IEF 5 3 Rehydration and sample application Fig 5 2 The Immobiline DryStrips Reswelling Tray Immobiline DryStrips holders can also be used Information on using Immobiline DryStrips holders can be found in the 2D Electrophoresis Principles and Methods handbook Protocol 1 Slide the protective lid completely off the Immobiline DryStrips Reswelling tray Ensure that the tray is clean and dry Level the tray by turning the levelling feet until the bubble in the spirit level is centred 2 C
89. r for Ettan DALT into the top of the buffer tank to the fill line 4 Program the desired run parameters into the control unit see Table 6 1 for recommendations Table 6 1 Recommended running conditions Step Run duration Effect per gel Temperature 1 45 min 2W 20 C 2 4h 17W 20 C Note The recommended running conditions are for runs including twelve gels and should only be used as guidelines In runs with fewer gels the electric effect can be increased up to a maximum of 20 W per gel which will reduce run times 5 Close the lid of the buffer tank and press start stop to begin electrophoresis 6 Run the gel until the bromophenol blue dye front reaches the bottom of the gel 56 Ettan DIGE System User Manual 18 1173 17 Edition AB Second dimension SDS PAGE 6 7 Scan the gels as soon as possible after the second dimension SDS PAGE is finished in order to minimize protein diffusion see chapter 7 Image acquisition Keep the gels between the glass plates Note Do not fix the gels before the gels are scanned as this may affect DeCyder 2D software quantitation of CyDye DIGE Fluor minimal dye labeled proteins 8 Ifyou cannot scan the gels immediately store the gels in SDS electrophoresis running buffer for Ettan DALT at 4 C in the dark and keep the gels moist However allow the gels to reach room temperature before scanning Gels scanned more than a day after running are likely to show significant diffusion of the p
90. rate highly reproducible first dimension isoelectric focusing results Up to twelve Immobiline DryStrips can be run at the same time Fig 5 1 PGphor 3 isoelectric focusing apparatus and IPGphor 3 Control software Ettan DIGE System User Manual 18 1173 17 Edition AB 41 5 First dimension isoelectric focusing IEF 5 2 Workflow 42 WARNING The use of Ettan IPGphor Isoelectric Focusing IEF System includes use of high voltage Read 2D Electrophoresis Principles and Methods Ettan IPGphor 3 Instrument User Manual and Ettan IPGphor 3 Safety Handbook prior to operation of the Ettan IPGphor IEF System for detailed instructions and safety information 5 1 1 General precautions for good results 2D analysis of protein samples is highly sensitive for contaminations and in order to achieve good and reproducible results some general precautions must be considered e Wear gloves to minimize protein contamination e Clean all components with IPGphor Strip Holder cleaning solution code no 80 6452 78 and Milli Q water to remove traces of protein before and after use e Ensure the Immobiline DryStrips do not dry out 5 2 Workflow 1 Rehydration of Immobiline DryStrips 2 Sample application to Immobiline DryStrips Two options described e Cup loading protocol e Rehydration loading protocol 3 IEF on Ettan IPGphor 3 apparatus 5 2 1 Sample application protocol selection The protocol to choose is dependent of th
91. rative G lS mum 9 2 SCS ONS CAEL OF g4sedrnsesss ironin ss sinnen arr 9 3 First dimension isoelectric focusing IEF 9 4 Second dimension SDS PAGE 9 4 1 Clean and Bind Silane treat the Glass plates ssssssssssseeieeeeeee 81 9 4 2 Attach reference MGFKELS massvis 83 9 4 3 Second dimension SDS PAGE mmm 84 9 5 Post staining with Deep Purple Total Protein Stain on 84 9G GELSIN enrera saa a aa A 85 9 7 Matching gels and creating a pick list ssmsmmmmmemssmm 86 98 SPOt PICKING the gel sssusa etisi 86 9 9 Mass spectrometry analysis w wmmmemsssmsessssssmmssmm 87 S M ROE E 01 E EEEE A E nde 87 Appendix A Testing cell lysates for successful labeling A 1 Testing new cell lysate for successful labellNQ susmmsmn 91 AL Recipes euina na E 96 Appendix B Labeling of cell surface proteins B 1 Selective labeling of cell surface PFOtOINS ssmmsmmsmm 97 Appendix C Reagents tested for compatibility with Ettan DIGE system C 1 List Of reagents wo eeeccccccccssssssssssssesssssssccssssssssssssesessessecsesssssssssuussssesscessesssssess 99 Appendix D Trouble shooting guide D 1 Sample preparation and labeling s ssmmmsmmemsesemmummmmmsm 103 D 2 First dimension ClECtrOPNOFESIS eeessccccsscssssssssseemesssssccsssssssssssseesesseeeee 105 D 3 Second dimension electrophoresis s sssmsmmmmsmsmummmmmsm 106 D 4 Typhoon Variable Mode Imager resu
92. rent gels This is performed by comparison of the ratios of sample standard rather than direct comparison of raw spot volumes In this way differences in spot intensity that may arise due to experimental factors during the process of 2D electrophoresis such as protein loss during sample transfer will be the same for each sample within a single gel including the internal standard This means that the relative ratio of sample standard will not be affected by such variation due to experimental factors Spot volume i e the sum of the pixel values within a spot minus background for each experimental sample is compared directly to the internal standard by DeCyder 2D Spot ratios are calculated volume of secondary image spot volume of primary image spot indicating the change in spot volume between the two images The protein abundance for each spot in each sample is then expressed as a normalized ratio relative to the internal standard the primary image e g Cy3 sample 1 Cy2 standard and Cy5 sample 2 Cy2 standard From this analysis cross sample comparisons can be made see Table 2 2 Ettan DIGE System User Manual 18 1173 17 Edition AB DIGE concepts 2 Table 2 2 Ratio of sample internal standard for a single protein of interest Sample Ratio of sample internal standard for a single protein of interest Al A2 A3 A4 B1 B2 B3 B4 2 1 2 4 21 9 2 5 2 6 25 2 2 2 4 No
93. rk is performed between 400 and 900 V The voltage chosen depends on the type and quantity of dye or stain present A quick prescan at 500 or 1 000 um pixel resolution should be performed to identify a suitable voltage This allows a rapid scan at a relatively low resolution that should not be used for quantitative analysis It does however give an approximation of expected signal values which will aid determination of the PMT voltage required The prescan can be opened in ImageQuant TL software Spots showing the most intense signal should be selected using one of the Object tools such as the rectangle Higher resolution 100 um scans must be used to collect quantitative data This resolution is required for subsequent data analysis using DeCyder 2D Using the Volume Review tool button displays the information associated with Ey the selected area in the format Inspector ix Volume Quantitation Statistics Maximum pixel value and X Y co ordinates Object Name Volume Background Average Std Dev Sum Median Area Perimeter Centroid Width Height Max Yal pos Min Vallpos Sum Above BG Pixels Above BG RECT 1 135208879 36 0 000 None 5671 513 9779 156 135208879 36 2205 112 23840 618 923 15 647 31 160 149 1 75295 36 934 654 787 48 854 693 135208879 357 23840 Note The maximum pixel value should not exceed 100 000 as this indicates signal saturation has been reached
94. rotein extraction y saturation dyes preparative vial 22 Se std idei it EA Cells Sample Perform first dimension electrophoresis 4 Ettan IPGphor 3 Isoelectric Focusing System Prepare the Ettan DALT gel Bind Silane treatment Attach reference markers e gt Cast the gel Glass plate Perform second dimension electrophoresis Ettan DALTtwelve or Ettan DALTsix electrophoresis system Post stain and scan the gel Stain the gel Scan the gel Preparative gel Post stained gel Scanned gel image Match the gels and create a pick list Matching gels Creating a pick list Pick list Proteins of interest found in the analytical workflow Matching ha mm oat Sao F a Pick gel stained preparative gel Preparative gel image Master image Spot pick the post stained gel MALDI slides Pick list i n txt Pick gel stained preparative gel Ettan Spot Picker Perform mass spectrometry analysis Mass spectrum MALDI slides gt Protein ID M Z Fig 9 1 Preparative gel workflow in Ettan DIGE System 80 Ettan DIGE System User Manual 18 1173 17 Edition AB Preparative workflow 9 9 2 Sample preparation Amount of sample Prepare samples as described in chapter 4 However on a preparative gel larger amounts of protein sample are usually loaded typically 500 ug or more P
95. rotein labeling Preparative gel samples do not require labeling with CyDye DIGE Fluor minimal dyes It is recommended to use Deep Purple for post staining instead see section 9 5 However if samples have been labeled with CyDye DIGE Fluor saturation dyes they can be used directly in preparative gels In CyDye DIGE Fluor Labeling Kit for Scarce Samples and Preparative Gel Labeling a vial of Cy3 dye is included which allows labeling of up to 500 ug of protein following the protocol 25 8009 83PL 9 3 First dimension isoelectric focusing IEF For preparative workflow first dimension isoelectric focusing follow protocols in chapter 5 considering e Use rehydration loading section 5 3 or paper bridge loading see 2D Electrophoresis Principles and Methods handbook protocols for sample application e The loading should be optimized for different strip lengths and pH ranges For a pH 3 10 NL 24cm Immobiline DryStrips up to 600 ug of protein can be loaded on a preparative gel 9 4 Second dimension SDS PAGE For preparative workflow second dimension SDS PAGE electrophoresis please consider e Clean and Bind Silane treat the glass plates to be used e Attach reference markers to the treated glass plates e Ensure correct orientation of Immobiline DryStrips and the preparative second dimension SDS PAGE gel 9 4 1 Clean and Bind Silane treat the glass plates The Bind Silane treatment of the glass plates is performed to immobilize the gel
96. rotein spots Ettan DIGE System User Manual 18 1173 17 Edition AB 57 6 Second dimension SDS PAGE 6 7 Recipes 6 7 Recipes Displacing solution Reagents Quantity Final concentration Tris 1 5 M pH 8 8 25 ml 375 mM Glycerol 87 v v 57 5 ml 50 v v 1 Bromophenol blue stock 200 ul 0 002 w v solution Make up to 100 ml with distilled water Prepare fresh and use immediately Do not store 12 5 Second dimension SDS PAGE gel composition for Ettan DALT Reagents Quantity for 900 ml of a 12 5 gel Acrylamide PAGE 40 w v 281 25 ml PlusOne Methylenebisacrylamide 150 3 ml 2 w v Tris 1 5 M pH 8 8 225 ml 10 w v SDS 9 0 ml 10 v v TEMED 1 24 ml 10 w v APS 9 0 ml Make up to 900 ml with distilled water e Use chilled solutions e Prior to addition of APS filter the solution through a 0 2 micron filter into a clean bottle and then degas the solution e Prepare fresh APS solution on day of use e Add the APS solution immediately prior to use to prevent polymerization in the bottle 58 Ettan DIGE System User Manual 18 1173 17 Edition AB Second dimension SDS PAGE 6 SDS electrophoresis running buffer for Ettan DALT Reagents Quantity Final concentration Tris MW 121 14 60 5 g 25mM Glycine MW 75 07 288g 192 mM SDS MW 288 38 40g 0 2 w v Make up to 20 I with distilled water Store at room temperature Stable for 3 months
97. s also reduced as half the amount of internal standard is used six gels instead of twelve gels 3 3 2 CyDye DIGE Fluor saturation dyes Comparison of protein abundance between three differently treated samples A C each with four biological replicates using e CyDye DIGE Fluor Cy3 and Cy5 saturation dyes e Internal standard labeled with CyDye DIGE Fluor Cy3 saturation dye e All experimental samples labeled with the same dye CyDye DIGE Fluor Cy5 saturation dye e Twelve gels are required loaded as described in Table 3 1 Ettan DIGE System User Manual 18 1173 17 Edition AB 27 3 Experimental design 3 3 Examples of experimental design Experimental set up e Mix 5 yg of each of the 12 samples A1 A4 B1 B4 and C1 C4 together to create 60 ug of the internal standard e Label the internal standard with CyDye DIGE Fluor Cy3 saturation dye e Individually label 5 ug of samples A1 A4 B1 B4 and C1 C4 with CyDye DIGE Fluor Cy5 saturation dye e Twelve gels are required loaded as described in Table 3 2 Table 3 2 Gels to be run for Case study 2 Samples labeled with CyDye DIGE Fluor saturation dyes Gel Cy3 Standard Cy5 1 5 ug 4 17ug each of A1 4 B1 4 C1 4 5 ug sample B4 2 5 ug 4 17ug each of A1 4 B1 4 C1 4 5 ug sample C2 3 5 ug 4 17ug each of A1 4 B1 4 C1 4 5 ug sample A4 4 5 ug 4 17ug each of A1 4 B1 4 C1 4 5 ug sample A2 5 5 ug 4 17ug each of A1 4 B1 4 C1
98. scontinue the product described at any time without notice or obligation Contact your local GE Healthcare representative for the most current information CyDuye 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE technology is covered by US patent numbers US6 043 025 US6 048 982 US6 127 134 and US6 426 190 and foreign equivalents and exclusively licensed from Carnegie Mellon University CyDuye This product or portions thereof is manufactured under licence from Carnegie Mellon University under US patent number US5 268 486 and other patents pending The purchase of CyDye fluors includes a limited license to use the CyDye fluors for internal research and development but not for any commercial purposes A license to use the CyDye fluors for commercial purposes is subject to a separate license agreement with GE Healthcare GE Healthcare has patent applications pending relating to its DeCyder software technology European patent application number EP1 234 280 2005 General Electric Company All rights reserved GE Healthcare Bio Sciences AB a General Electric company GE Healthcare Bio Sciences AB Bjorkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthc
99. sed Immobiline DryStrips 4 Second dimension SDS PAGE 6 3 Casting homogeneous gels Note Prepare gels at least one day before use to ensure reproducible results 1 Use low fluorescence glass plates without scratches in order to achieve high quality images with low background 2 Ensure the entire gel casting system is clean dry and free of any polymerized acrylamide 3 Prepare 100 ml of displacing solution 4 Prepare acrylamide gel stock solution For a full 14 gel set make up 900 ml of solution Add TEMED but do not add APS This amount of gel solution will provide sufficient volume to cast gels using either a funnel or a peristaltic pump Tip Use chilled gel solutions This will increase reproducibility since temperature effects are minimized WARNING Acrylamide is a neurotoxin Never pipette by mouth and always wear protective gloves when working with acrylamide solutions Immobiline DryStrips or surfaces that come into contact with acrylamide solutions 5 Remove dust by filtering the acrylamide gel stock solution with a standard bottle top filter apparatus Dust may create fluorescent artefactic dots on images 6 Degas the acrylamide gel stock solution using a vacuum pump 7 Assemble the gel caster as described in the Ettan DALT electrophoresis unit user manuals The caster should be placed on a level bench or on a levelling table so that the gel tops are level Note Do not apply excess of filter or separat
100. t focused Poor electrical contact in first dimension Check that the strips are in contact with the strip holder electrodes Ettan DIGE System User Manual 18 1173 17 Edition AB 105 D Trouble shooting guide D 3 Second dimension electrophoresis D 3 Second dimension electrophoresis Problem Cause Remedy No protein spots are visible on the gel Incorrect labeling protocol Check the sample preparation and labeling protocol Inefficient sample solubilization Increase concentration of solubilizing components in the sample solution The upper concentration limits for common reagents are Urea 9 8 M Thiourea 2 M zwitterionic detergent CHAPS 4 Pharmalyte 1 DTT 2 3 No or insufficient SDS in electrophoresis running buffer Ensure electrophoresis running buffer is correctly formulated Individual spots appear as multiple bands or are missing unclear or inthe wrong position Immobiline DryStrips placement Ensure that the plastic backing of the Immobiline DryStrips are against the glass plate on the second dimension gel directly onto the top of the acrylamide gel Protein oxidation during electrophoresis Prevent oxidation of oxygen sensitive proteins in the gel Check the correct equilibration conditions are used prior to the second dimension separation DTT reduction then treatment with iodoacetamide alkylates the sulphydryl groups and thus prevents the reduced
101. te polyacrylamide gel electrophoresis SDS PAGE Both systems are designed for simple assembly and rapid electrophoresis Ettan DALTsix accepts up to six large second dimension SDS PAGE gels and Ettan DALTtwelve accepts up to twelve large second dimension SDS PAGE gels The Ettan DALT electrophoresis systems are designed to handle multiple large format second dimension SDS PAGE gels in a simple efficient and reproducible manner Ettan DALT gels are large enough to accommodate the longest Immobiline DryStrips 24 cm For Ettan DIGE system applications low fluorescent glass plates must be used These glass plates reduce background signal and will therefore improve the quality of results Ettan DALT gels are poured using a gel caster Up to 14 Ettan DALT gels can be prepared in a single batch For detailed description of Ettan DALT electrophoresis systems Ettan DALTsix and Ettan DALTtwelve components for preparation of Ettan DALT gels and for detailed protocols for loading gels into the caster and casting Ettan DALT gels consult the Ettan DALTtwelve system user manual and the Ettan DALTsix system user manual Fig 6 1 Ettan DALTsix left and Ettan DALTtwelve right electrophoresis systems Ettan DIGE System User Manual 18 1173 17 Edition AB 51 6 Second dimension SDS PAGE 6 2 Workflow 52 6 2 Workflow 1 Cast homogeneous second dimension SDS PAGE gels 2 Equilibration of focused Immobiline DryStrips 3 Loading of focu
102. te Down regulation of protein abundance relative to the internal standard is denoted by a negative prefix for example a two fold decrease or a conventional ratio of 0 5 is displayed as 2 0 DeCyder 2D software can graphically display the relative abundance of each protein against the normalized internal standard see Fig 2 6 Standard Abundance Internal standard Hav 24 samples A1 4 samples B1 4 Fig 2 6 Plot of sample ratios relative to normalized internal standards Ettan DIGE System User Manual 18 1173 17 Edition AB 23 2 DIGE concepts 2 6 Statistical tests of protein abundance in DeCyder 2D 24 It is possible to compare the protein abundance for a protein of interest in different samples The ratio of sample A sample B for the protein of interest shown in Table 2 2 and Fig 2 6 can be calculated see Table 2 3 The result shows that the protein of interest is down regulated approximately five fold in sample A compared to sample B Table 2 3 Ratio of sample A sample B calculated from sample standard ratios shown in Fig 2 6 Sample Ratio of sample A sample B A1 B1 5 4 A2 B2 6 0 A3 B3 4 2 A4 B4 6 0 2 6 Statistical tests of protein abundance in DeCyder 2D Statistical tests are important and give the user a level of confidence by taking into account the inherent biological variation within a group compared to the induced difference between groups and assigning a confidence rat
103. to use However gels can be stored in an airtight container at 4 C for up to one week provided they are submersed in 375 mM Tris HCl to keep the gels from drying out Ettan DIGE System User Manual 18 1173 17 Edition AB 53 6 Second dimension SDS PAGE 6 4 Equilibration of focused Immobiline DryStrips 6 4 Equilibration of focused Immobiline DryStrips Note Equilibrate focused strips immediately before the second dimension SDS PAGE run 1 Prepare SDS equilibration solutions 1 and 2 Allow 10 ml per strip for each equilibration solution 2 With forceps carefully remove the Immobiline DryStrips from the IPGphor Cup Loading Strip Holder If the Immobiline DryStrips have been focused and stored frozen allow the strips to defrost completely beforehand 3 Place the Immobiline DryStrips in individual equilibration tubes code no 80 6467 79 with the support film toward the wall 4 Add 10 ml of the DTT containing equilibration solution 1 to each tube 5 Incubate the strips for 15 min with gentle agitation Do not over equilibrate as proteins can diffuse out of the strip during this step 6 During equilibration prepare the gel cassettes for loading by rinsing the top of the gels with running buffer Place the glass plates in the rack upside down 7 For samples labeled with CyDye DIGE fluor minimal dyes Pour off the equilibration solution 1 and add 10 ml of equilibration solution 2 Incubate the strips for 15 min with gentle agitation
104. tock solution to 3 ul DMF 3 Ensure all dye is removed from the pipette tip by pipetting up and down the working dye solution several times Ettan DIGE System User Manual 18 1173 17 Edition AB 35 4 Sample preparation and labeling 4 7 Labeling 4 7 3 CyDye DIGE Fluor minimal dye labeling The dye labeling reaction is designed to be simple and should take about 45 min to perform Recommended conditions e tis very important that the cell lysate protein sample has pH 8 5 in order to achieve an efficient labeling see section 4 4 e Dye to protein ratio of 400 pmol dye 50 ug protein If the ratio of dye protein is too low sensitivity may be decreased and if the ratio of dye protein is too high there is a possibility of multiple dye molecules per protein and this could lead to multiple spots per protein on the gel e Protein concentration of 5 10 mg ml in the cell lysate However samples containing 1 mg ml protein have been successfully labeled using the protocol below Protocol The protocol illustrates labeling of a cell lysate using 400 pmol of dye to label 50 ug of protein 1 Add a volume of protein sample equivalent to 50 ug to a microfuge tube Bulk labeling reactions can be performed by scaling up as required 2 Add 1 ul of working dye solution 400 pmol to the microfuge tube containing the protein sample 3 Mix dye and protein sample thoroughly by pipetting and vortexing 4 Centrifuge briefly in a microcentrifu
105. ty check box It is essential that this is selected for analytical gels to ensure that all scans are carried out as individual scans g Click OK 66 Ettan DIGE System User Manual 18 1173 17 Edition AB Image acquisition 7 4 Select orientation orienting options button labeled 4 in Fig 7 2 see section 7 4 step 5 Options H Orientation es x R YH bmple Pixel size M AL IE 100 microns 3 mm Tip To aid selection of the correct character the Gel Orientation Guide can be overlaid above the gel once it is in position on the Typhoon platen The appearance of the overlaid letter R on the Gel Orientation Guide indicates which character to select on the Typhoon Scanner Control software screen Note ImageQuant TL Tools can be used to flip or rotate images which were scanned in wrong orientation 5 Select the Press Sample option labeled 5 in Fig 7 2 if scanning gels between glass plates CAUTION The Press Sample feature should not be used with naked or backed gels as it can damage the instrument 6 Select Pixel size from the list labeled 6 in Fig 7 2 A quick pre scan at 1000 um pixel resolution should be performed initially to identify a suitable PMT voltage e Select 100 um as pixel size for DALT gels e Select 25 or 50 um as pixel size for minigels 7 Select Focal Plane from the list labeled 7 in Fig 7 2 The 3 mm setting is used for most applications of the Ettan DIGE system
106. type of enrichment partly due to their low abundance 1 2 of total cellular protein contents They are often poorly represented in second dimension SDS PAGE gels due to their hydrophobic nature and high molecular weight The new protocol presented in this appendix for selective labeling of cell surface proteins using CyDye DIGE Fluor minimal dyes makes it is possible to visually enrich cell surface proteins without performing sample fractionation This protocol is fast simple to use and all three CyDye DIGE Fluor minimal dyes can be used to label cell surface proteins These features allow for multiplexing using Ettan DIGE technology and analysis of protein expression using DeCyder 2D software In this way the level of surface proteins can be studied in different disease states or when responding to different treatments Small changes in abundance can be detected with high accuracy and results are supported by defined statistical methods Protocol 1 Grow the cells of interest until a minimal cell number of 5 10 x 10 cells confluent or cells in suspension is reached For cells growing in suspension proceed to step 3 2 Carefully detach adherent cells non enzymatically cell dissociation media enzyme free PVS based 3 Count the cells and pellet them by centrifugation From now on keep the cells on ice 4 Wash the cells by resuspending the pellet in 1 ml cold HBSS pH 8 5 buffer and transfer to 1 5 ml eppendorff tube Centrifuge
107. ubbles under the Immobiline DryStrips 4 Overlay each Immobiline DryStrips with PlusOne DryStrips Cover Fluid to prevent evaporation and urea crystallization 5 Slide the lid onto the Immobiline DryStrips Reswelling Tray and allow the Immobiline DryStrips to rehydrate at room temperature A minimum of 10 his required for rehydration overnight is recommended Tip Strips can be rehydrated under low voltage 30 50V when using the rehydration loading protocol 5 3 2 Preparations for first dimension run including Cup loading After rehydration Immobiline DryStrips are prepared for first dimension isoelectric focusing Samples are loaded according to the cup loading protocol in the Manifold Ettan DIGE System User Manual 18 1173 17 Edition AB 45 5 First dimension isoelectric focusing IEF 5 3 Rehydration and sample application Protocol 1 Place precut electrode papers on a clean dry surface such as a glass plate and soak with deionized water Remove excess water by blotting with filter paper Note Itis important that the paper electrodes are damp and not wet Excess water may cause streaking Transfer the Immobiline DryStrips from the Reswelling Tray to the Manifold by using a pair of forceps Place the Immobiline DryStrips with gel side up and with the acidic end of the strips oriented toward the anodic side of the instrument Place a damp paper electrode from step 1 onto the acidic and basic ends of the gel
108. uccessful labeling 11 Insert the gel into the scanner in the correct orientation see Chapter 7 Image acquisition f H ee H Hoo a E as 25 125 6 25 25 12 5 6 25 25 12 5 6 25 25 12 5 6 25 VV RR Sample 1 Sample 2 Sample 3 Control ug protein Fig A 1 CyDye DIGE Fluor Cy5 minimal dye scanned image 12 Quantify the labeling of each protein sample using ImageQuant TL software 92 Ettan DIGE System User Manual 18 1173 17 Edition AB Testing cell lysates for successful labeling A 13 Carry out the statistics by opening the image in ImageQuant TL software Draw a single box over the first lane using the Object Rectangle Tool Copy and Paste the rectangle for all of the samples that need to be tested in the remaining lanes t ji d Ile eee RP Rate S lang sleet Mm leer off ea ie i ai wai i ii ee as 25 125 625 25 125 625 25 125 625 25 125 625 Save Ses Sample 1 Sample 2 Sample 3 Control ug protein Fig A 2 CyDye DIGE Fluor Cy5 minimal dye scanned image Lanes are overlaid with identical boxes to give a volume report in ImageQuant TL Toolbox Ettan DIGE System User Manual 18 1173 17 Edition AB 93 A Testing cell lysates for successful labeling A 1 Testing new cell lysate for successful labeling 14 In Analysis Volume Report Setup highlight the boxes Object Name Volume Area and select Results Only in the Print Format section Yolume Report Setup d xj r Header
109. up loading protocol Pipette the appropriate volume of rehydration solution or DeStreak Rehydration solution into each slot to be used see Table 5 2 Deliver the solution slowly along the slot Remove any large bubbles For complete sample uptake do not apply excess rehydration solution Note Use DeStreak Rehydration Solution to reduce streaking especially in the pH range 7 11 DeStreak Rehydration Solution contains DeStreak Reagent that prevents unspecific oxidation of protein thiol groups during electrophoresis Rehydration loading protocol Use labeled protein samples prepared as described in section 4 8 The total volume must not exceed the stated values in Table 5 2 If the volume is larger the sample must be split or concentrated Pipette the appropriate volume of sample into each slot to be used Deliver the solution slowly along the slot Remove any large bubbles 44 Ettan DIGE System User Manual 18 1173 17 Edition AB First dimension isoelectric focusing IEF 5 Table 5 2 Rehydration volumes of Immobiline DryStrips Immobiline DryStrips length cm Total volume per strip ul including sample 7 125 11 200 13 250 18 340 24 450 3 Remove the protective cover foil from the Immobiline DryStrips gel Use forceps to position the strip with the gel side down To help coat the entire strip gently lift and lower the strip and slide it back and forth along the surface of the solution Be careful not to trap b
110. with randomized sample labeling of either CyDye DIGE Fluor Cy3 or Cy5 minimal dyes is strongly recommended Samples to be evenly distributed between the CyDye DIGE Fluors Cy3 and Cy5 and between gels Experimental set up e Mix 50 ug of each of the 12 samples A1 A4 B1 B4 and C1 C4 together to create 600 ug of the internal standard and label with CyDye DIGE Fluor Cy2 minimal dye e Individually label 50ug of samples A1 A4 B1 B4 and C1 C4 with either CyDye DIGE Fluor Cy3 or Cy5 minimal dye in a randomized design e Six gels are required loaded as described in Table 3 1 Ettan DIGE System User Manual 18 1173 17 Edition AB Experimental design 3 Table 3 1 Gels to be run for Case study 1 Samples labeled with CyDye DIGE Fluor minimal dyes Gel Cy2 Internal standard Cy3 Cy5 1 50 ug 4 17ug each o 50 ug sample B2 50 ug sample C1 samples A1 4 B1 4 C1 4 2 50 ug 4 17ug each o 50 ug sample A1 50 ug sample B3 samples A1 4 B1 4 C1 4 3 50 ug 4 17ug each o 50 ug sample C3 50 ug sample A4 samples A1 4 B1 4 C1 4 4 50 ug 4 17ug each o 50 ug sample A3 50 ug sample C2 samples A1 4 B1 4 C1 4 5 50 ug 4 17ug each o 50 ug sample B4 50 ug sample A2 samples A1 4 B1 4 C1 4 6 50 ug 4 17ug each o 50 ug sample C4 50 ug sample B1 samples A1 4 B1 4 C1 4 Total gels 6 Note By using three dyes instead of two the number of gels required is halved The amount of material required i
111. y and advanced protocol handling The software provides a number of optimized protocols for running first dimension isoelectric focusing It is also possible to create new protocols import existing protocols and edit protocols During the run current and voltage can be monitored displayed in a graph and a log file created Up to four different instruments can be monitored from one PC software See Ettan IPGphor 3 Control Software User Manual for details Protocol using Ettan IPGphor 3 Control software 1 Start the Ettan IPGphor 3 Control software and turn on the Ettan IPGphor 3 instrument 2 Connect the software with the IPGphor 3 instrument on which the run is to be made 3 Select protocol for first dimension isoelectric focusing For selection of optimized protocols choose Fast mode protocol selection and enter data of experimental parameters Alternatively set protocol using the Advanced mode protocol selection Note Do not programme the Ettan IPGphor IEF unit to deliver more than 75 LA per Immobiline DryStrips 4 Start run by clicking the start button in the Ettan IPGphor 3 Control software The selected protocol is now downloaded to the selected instrument and the run is started 5 Ifthe Immobiline DryStrips are not run immediately on the second dimension gel they can be stored at 70 C in a sealed container The container has to be rigid because frozen Immobiline DryStrips are very brittle and can easily be damaged
112. yDye DIGE Fluor saturation dye solution Note Label the pooled protein internal standard sample with Cy3 and the experimental protein sample with Cy5 7 Mix vigorously by pipetting and spin 8 Incubate at 37 C for 30 min in the dark 9 Stop the reaction by adding an equal volume of 2x sample buffer 10 Mix vigorously by pipetting and spin Labeling is now finished The labeled samples can be processed immediately or stored for 1 month at 70 C in the dark Ettan DIGE System User Manual 18 1173 17 Edition AB 37 4 Sample preparation and labeling 4 8 First dimension sample preparation 4 8 First dimension sample preparation The main difference between conventional 2D electrophoresis and Ettan DIGE system is that the latter will enable up to three different protein samples to be run ona single 2D gel To achieve this you need to mix the differently labeled protein samples BEFORE the first dimension run Protocol It is recommended that 50 pg for CyDye DIGE Fluor minimal dyes or 5 ug for CyDye DIGE Fluor saturation dyes of each labeled protein sample is combined for each gel 1 Combine the two for CyDye DIGE Fluor saturation dyes or three for CyDye DIGE Fluor minimal dyes labeled samples into a single microfuge tube and mix One of these samples should be the pooled internal standard 2 For CyDye DIGE Fluor minimal dye labeled samples Add an equal volume of 2x sample buffer to the labeled protein samples and leave on ice

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