Antibody Microarrays 380 & 500 User Manual
Contents
1. ecsecsesesescsseceecscseseeeceesesescsaescseeceecscssacsesarseeesesavseaseasatecntaes 12 D Labeling Protein with Fluorescent Dye ccececssesescsssseecseseeeeecsesescscearscseaseecsensacesarseeesesasaeaeeasatecataes 13 E Removing Unbound Dye using PD 10 Desalting Columns eeseeesseseeesenseeeeeeeseeseeaeseseeeateeneees 14 F Determining Protein Concentration cacaeiiincinicagaconemuna craemtentetia diay ieeatdannngn 16 G Estimating the Average Number of Coupled Dye Molecules ceccsecstessesessseeesneesees 17 VII Protein Extraction amp Labeling Small Scale Protocol c cccssccssceesssseesseeeesseeesseeees 19 A Extracting Protein from Crude Tissue cca cc uesie cui cueealie ie eeeea een 19 B Extracting Protein from Cultured Cells ccccsscasstineneotiaasieceuissceersanienninciainnaaeute 20 C Labeling Protein with Fluorescent Dye ssessecsscssssessncseseenseesesensesssecensessncesoesnsnessssssnesseeeseeassseso 21 D Removing Unbound Dye using Protein Desalting Spin Columns 0 ccc eesseeeeeeeseeeseeseseneneeaeeeneaes 22 E Determining Protein Concentration aiiccvcnuanmiunusnnniananinaiuigawudieninanuas 24 E Estimating the Average Number of Coupled Dye Molecules 0 ccecsseceesseeeseeeesneesnee 25 VIII Antibody Array Incubation Protocol ceccceseeceeee cee ee eee ee teense seaaeeeaeeeeeeeeeseseseeseaaneeeeeees 27 IX Analysis Of Results sensisse REE Saat NEEE KERET 31 X References icccccsccissc2. Use 20 ug of protein per slide J N 10 ug of Cy3 labeled protein and 10 ug of Cy5 labeled protein M Y Larger amounts of protein will saturate the antibodies and relevant results will not be Incubation Tray obtained For serum samples use Incubation Wash Incubation Wash 100 ug of protein since a large l l Step 4 percentage 80 of the protein Slide 1 Chambers Slide 2 Chambers Microarray incubation consists of immunoglobin and 1 5 hr albumin a Incubate tray at room temperature for 10 min and wash microarray slides in the provided storage vial to remove storage buffer as described in User Manual b Place microarray slides array side up in the incubation chambers using forceps or gloved hands Incubation Tray Incubation Wash re wast Slide 1 Chambers Slide 2 Chambers Incubate at room temperature for 40 min with constant rocking motion Transfer slides to their respective Drying amp Scanning Slides wash chambers and begin washing When drying slides place the slides in the vial with the array end up Do not touch the array surface at any time Dry slides by centrifugation Slides should be scanned within 24 hr after drying Slide pairs Step 5 should be scanned using the same Scanning microarrays scanner settings Scan 30 min Figure 1B The Ab Microarray procedure Steps 4 5 Clontech Laboratories Inc www clontech com Protocol No PT3648 1 ATakar
3. User Manual Antibody Microarrays 380 amp 500 User Manual EE Ss Clontech Cat Nos 631790 631795 631786 United States Canad 200 662 2566 631796 Asia Pacific 631797 1 650 919 7300 Europe 631798 33 0 1 3904 6880 PT3648 1 PR123821 oe oTielenk Published February 2011 Clontech Laboratories Inc ATakara Bio Company 1290 Terra Bella Ave Mountain View CA 94043 Technical Support US E mail tech clontech com www clontech com Antibody Microarrays 380 amp 500 User Manual Table of Contents l Introductio i iscisscvssszescccsisscasccnsasi svonseas saan eseadattestundias sbaenecin coi bevtiatisonsdkat teamed aaea iaraa 3 Il Protocol Overview amp Troubleshooting Guide cccccccecsseeeeeeeesseeeeeenseeeeeeeenseeeeeeeneneaes 4 Iil List of Componenta sssrinin rrenean EERE IEEE TENE EENEI EEE ETATER 6 IV Additional Materials amp Equipment Required cccccccccecsseeeeeeeesseeeeeeensseeeeeeesseeeeeeeneneaes 8 V Important Pre Protocol Considerations ccceceeeeeeeeeeeeeeeeeeeeeeeeeeeseeeeeseaaeeeeeeeeeeeeeeee 9 VI Protein Extraction amp Labeling Large Scale Protocol cccccsssscssseesssseesseeeeseeessneees 10 A Extracting Protein from Crude Tissue sssssseseesisessssssettrsrsssssstertrsrssssteterrrsssssesterrrsrsssssenert 10 B Extracting Protein from Cultured Cells centacnned octtauiasarinmeumnunnnnaeianaonr 11 C Preparing Protein from Body Fluids 0 0
4. 2 ab Array i Ab List y Import amp Analyses d Figure 2 The Ab Microarray Analysis Workbook contains four worksheets The Ab Array and Ab List worksheets contain array specific information such as the names and coordinates of antibodies and the Locus Link and SWISS PROT accession numbers of the corresponding protein targets The fourth worksheet Import amp Analyses contains formulas that perform arithmetic operations on the fluores cence data i e Cy5 Cy3 signal ratios that you paste into the worksheet Other formulas in this sheet combine the values of these operations to generate an INR for each coordinate on the array The INR can be represented by the following expression INR Ratio 1 Ratio 2 where Ratio 1 A Cy5 relative fluorescence units B Cy3 relative fluorescence units and Ratio 2 B Cy5 relative fluorescence units A Cy3 relative fluorescence units Note that the Ratio 1 values are obtained from Slide 1 Whereas the Ratio 2 values are obtained from Slide 2 3 Click on the Import amp Analyses tab to make it the active window 4 Paste the Cy5 Cy3 signal ratios from each array into the appropriate columns of the worksheet Figure 3 Be sure that your Cy5 Cy3 ratios are listed in the same order as the corresponding Ab Ag in the worksheet When you paste your data into the worksheet it automatically calculates Ratio 1 Ratio 2 and places these values in the next column which
5. 4 Std 0 5 Proteinblank Protein blank Protein blank Protein blank mg ml mg ml mg ml mg ml mg ml mg ml mg ml mg ml Sample A Cy3 Sample A Cy5 Sample B Cy3 Sample B Cy5 Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample ACy3 ACy3 ACy3 ACyS ACy5 ACy5 BCy3 BCy3 BCy3 B Cy5 B Cy5 B Cy5 Protocol No PT3648 1 www clontech com Clontech Laboratories Inc Version No PR123821 ATakara Bio Company 16 Antibody Microarrays 380 amp 500 User Manual VI Protein Extraction amp Labeling Large Scale Protocol continued 4 Calculate protein concentration AOD Sample OD protein sample OD protein blank 562 562 AOD Std OD standard OD blank 0 mg ml 562 562 Use the AOD and your standard curve to estimate protein concentration Typical concentration of desalted samples 0 2 mg protein ml P G Estimating the Average Number of Coupled Dye Molecules Po 1 Measure absorbance using a spectrophotometer Dilute sample with 1X Desalting Buffer 1 10 dilution Prepare blank with 1X Desalting Buffer Measure Cy3 absorbance at 552 nm can use 1 cm cuvettes Blank 1X Desalting Buffer Sample A Cy3 1 10 dilution in Desalting Buffer Sample B Cy3 1 10 dilution in Desalting Buffer Measure Cy5 absorbance at 650 nm can use 1 cm cuvettes Blank 1X Desalting Buffer Sample A Cy5 1 10 dilution in Desalting Buffer Sample B Cy5 1 10 dilution in Desalting Buffer Record A a
6. A Cy3 A Cy5 B Cy3 B Cy5 Small Scale Labeling User Manual Final labeled protein concentration should be 0 8 0 9 mg ml using Protein Desalting Spin Columns If the above concentrations are not obtained DO NOT proceed to the next step 100 ug protein 100 ug 100 ug E Mix 1 A Cy5 B Cy3 100 ug protein Measure the Substitution Degree If it is higher than 4 then relabel with diluted amounts of Cy dye Since the final labeling reaction volume must remain the same it is necessary to dilute fresh Cy dyes with larger volumes of the Extraction Labeling Buffer and to add the same 50 pl volume of diluted dye per labeling reaction y Mix 2 A Cy3 B Cy5 Ab Microarray Incubation Step1 Protein extraction 1 2 hr Step 2 Protein labeling 2 3 hr Step 3 Removin unbound dye 1 hr Figure 1A The Ab Microarray procedure Steps 1 3 The volumes and quantities given in Steps 1 and 2 correspond to those needed for the Large Scale Protein Extraction amp Labeling procedure Steps 4 amp 5 are shown in Figure 1B Protocol No PT3648 1 Version No PR123821 4 www clontech com Clontech Laboratories Inc ATakara Bio Company Antibody Microarrays 380 amp 500 User Manual ll Protocol Overview amp Troubleshooting Guide continued Ab Microarray Incubation Mix 1 Mix 2 20 ug protein 20 ug protein Recommended Sample Amounts 5 ml Incubation Buffer
7. E Protein concentration in sample BCA assay result average molecular weight x 10 0 18 60 000 x 10 BUM Dye protein Cy3 concentration in sample protein concentration in sample RIM uM 2 For best results the dye protein ratio should be in the range of 2 4 When this ratio is significantly greater e g gt 6 the label may begin to interfere with antigen antibody binding 4 Sample calculation Sample A Cy5 Sample data BCA assay results protein concentration by BCA 0 18 mg ml 1 5 Absorbance results A Sample calculation Cy5 concentration in sample A so Esso x 10 0 9 150 000 x 10 6 uM Protein concentration in sample BCA assay result average molecular weight x 10 0 18 60 000 x 10 SMI Dye protein Cy5 concentration in sample protein concentration in sample SM 3M 2 For best results the dye protein ratio should be in the range of 2 4 When this ratio is significantly greater e g gt 6 the label may begin to interfere with antigen antibody binding Protocol No PT3648 1 www clontech com Clontech Laboratories Inc Version No PR123821 ATakara Bio Company 26 Antibody Microarrays 380 amp 500 User Manual Vill Antibody Array Incubation Protocol Clontech Laboratories Inc ATakara Bio Company 1 Prepare Incubation Buffer and set up Incubation Tray Prepare Incubation Buffer 45 ml Mix 4 5 ml Background Reducer amp 40
8. First Annual Great Lakes Bioinformatics Retreat Michigan USA de Wildt R M Mundy C R Gorick B D amp Tomlinson I M 2000 Antibody arrays for high throughput screening of antibody antigen interactions Nat Biotechnol 18 989 994 Venter J C et al 2001 The sequence of the human genome Science 291 1304 1351 Yamagiwa Y Marienfeld C Meng F Holcik M amp Patel T 2004 Translational regulation of X linked inhibitor of apoptosis protein by interleukin 6 a novel mechanism of tumor cell survival Cancer Res 64 1293 1298 Zhou H Roy S Schulman H amp Natan M J 2001 Solution and chip arrays in protein profiling Trends Biotechnol 19 suppl S34 S39 Protocol No PT3648 1 Version No 34 PR123821 www clontech com Clontech Laboratories Inc ATakara Bio Company Notes Antibody Microarrays 380 amp 500 User Manual Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnos tic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories Inc Milli Q is a registered trademark of Millipore Corporation Microsoft is a registered trademark of Microsoft
9. can use 1 cm cuvettes Blank 1X Desalting Buffer Sample A Cy3 1 10 dilution in Desalting Buffer Sample B Cy3 1 10 dilution in Desalting Buffer Measure Cy5 absorbance at 650 nm can use 1 cm cuvettes Blank 1X Desalting Buffer Sample A Cy5 1 10 dilution in Desalting Buffer Sample B Cy5 1 10 dilution in Desalting Buffer Record A and Aso for all samples A s Sample A Cy3 absorbance 552 nm of Sample A Cy3 absorbance 552 nm of blank A s Sample B Cy3 absorbance 552 nm of Sample B Cy3 absorbance 552 nm of blank A Sample A Cy5 absorbance 650 nm of Sample A Cy5 absorbance 650 nm of blank A Sample B Cy5 absorbance 650 nm of Sample B Cy5 absorbance 650 nm of blank 2 General considerations Assume that the average molecular weight of protein is 60 000 Da Protein concentration use the values obtained from the BCA method Molar extension coefficient lt 6552 of Cy3 150 000 M7 cm 650 of Cy5 250 000 M cm Clontech Laboratories Inc www clontech com Protocol No PT3648 1 ATakara Bio Company Version No PR123821 25 Antibody Microarrays 380 amp 500 User Manual Vil Protein Extraction amp Labeling Small Scale Protocol continued 3 Sample calculation Sample A Cy3 Sample data BCA assay results protein concentration by BCA 0 18 mg ml Absorbance results A 0 9 s52 Sample calculation Cy3 concentration in sample A x 10 0 9 150 000 x 10
10. in Figure 3 is labeled R R IMPORT Slide 41 Slide 12 Ratio of Ratios Slide 1 SlideH2 Figure 3 The Import amp Analyses worksheet has three sections Import Analyses and Sorting To use this worksheet first paste your fluorescence data into the Import section shown Be sure the data correspond to the correct antibody antigen Ab Ag pairs given in the leftmost column Note The view shown is that from the Ab Microarray Workbook 5 Choose File gt Save As and save a copy of the workbook under a new name 6 Copy the data in the Ab Ag Average R R and Average INR columns Clontech Laboratories Inc Protocol No PT3648 1 www clontech com ATakara Bio Company Version No PR123821 32 Antibody Microarrays 380 amp 500 User Manual IX Analysis of Results continued 7 Paste the data into the corresponding columns in the Sorting section of the worksheet Figure 4 SORTING Copy the data in columns E F and G row 6 517 Paste as Step 3 values in columns H I Ab Ag and J from row 6 and sort by Average INR column J Average R R Average INR Figure 4 The Sorting section of the Import amp Analyses worksheet 8 Choose Data gt Sort and sort the data by Average INR in ascending or descending order Interpretation of Results Average values e g Average R R and Average INR are usually considered invalid if they are based on duplicates that differ b
11. of proteins both cytosolic and membrane bound repre senting a broad range of biological functions The Ab Microarray 380 Disease Profiling Array enables you to detect many different proteins that are known to be associated with a variety of conditions such as cancer metastasis multiple sclerosis Parkinson s Alzheimer s obesity transplanted organ rejection digestive disorders and more Examples of the Ab Microarray s utility include the study of expression of apoptosis regulatory proteins Yamagiwa et al 2004 as well as the study of expression of proteins relevant to diabetes Gosmanov et al 2004 This array allows you to detect differences in protein abundance between two individual samples cells whole tissue or biological fluids The fluorescence based procedure which takes less than a day to complete lets you detect as little as 20 pg ml of each protein target A dual color detection method is uniquely designed so that inherent variations in dye labeling do not affect the outcome of the experiment Thus you can be confident that your side by side comparisons reflect the rela tive abundances of proteins in the sample Follow up studies with Western blotting and in situ hybridization support these claims Song et al 2002 Our proprietary process ensures that antibodies remain functionally active even after they are covalently immobilized to the glass surface The arrays are printed on 75 x 25 x 1 mm glass slides an open platf
12. the original extract in a 2 ml tube If the volume exceeds the tube s capacity use a second 2 ml tube Centrifuge suspension 10 000 x g for 30 min Transfer the supernatant to a prechilled 15 ml conical centrifuge tube Do not disturb the pellet Mix lysate by gently inverting the tube www clontech com Clontech Laboratories Inc ATakara Bio Company Antibody Microarrays 380 amp 500 User Manual VI Protein Extraction amp Labeling Large Scale Protocol continued 3 Measure protein concentration amp dilute sample Measure protein concentration Use Pierce s BCA Protein Assay Reagent Kit Dilute sample with Extraction Labeling Buffer 1 1 mg protein ml Final volume must be 21 mi Proceed immediately to Section D If final volume is below 1 ml STOP Do not proceed to the next step Repeat the extrac tion with a fresh sample or carry out small scale labeling starting at Section VII C P B Extracting Protein from Cultured Cells Protocol Note For adherent cells that are 90 confluent we find that two 150 mm culture plates when combined yield 150 mg of cells We typically harvest two 150 mm plates for each Sample A and B Before starting the freeze thaw procedure we wash the cells four times with PBS 20 volumes each wash 1 Cell preparation Cultured cells 50 150 mg Centrifuge in a preweighed centrifuge tube Decant the supernatant Aspirate the residual liquid Centrifuge the tube again for 2 min Aspirat
13. water completely Dry the slides as follows Place the slides array end up in the empty green capped Storage Vial provided amp cap it Centrifuge the slides for 5 min 1 000 x g at RT Using gloved hands uncap the vial While holding your finger over the top of the vial to prevent the slides from falling out tip the vial slightly to nudge the slides near the rim of the vial When the slides protrude by 2 cm remove the slides one by one The slides are now ready for scanning see Step 7 Clontech Laboratories Inc www clontech com Protocol No PT3648 1 ATakara Bio Company Version No PR123821 29 Antibody Microarrays 380 amp 500 User Manual Vill Antibody Array Incubation Protocol continued 7 Scan the Ab Microarray Slides with a Microarray Scanner Important If you need to postpone the scanning keep the slides in a dry chamber and protect them from light until you are ready to scan Scanning Arrays e Slides should be scanned lt 24 hours after drying e We routinely scan the arrays with an Axon GenePix 4000B scanner using the following settings e 635 nm Cy5 channel PMT 670 V power 33 e 532 nm Cy3 channel PMT 550 V power 33 e The bovine serum albumin control spots can be used as a guide for setting the scanner These control spots should generally give a fluorescent signal of 2 500 30 000 fluorescence units FU If the control spots are gt 50 000 FU this is an indication that the scanner se
14. 0 mm culture plate yields 17 mg of cells Before starting the freeze thaw procedure we wash the cells four times with PBS 20 volumes each wash 1 Cell preparation Cultured cells 15 25 mg Centrifuge in a preweighed microcentrifuge tube Decant the supernatant Aspirate the residual liquid Centrifuge the tube again for 2 min Aspirate any residual traces of liquid Weigh the cell pellet Reweigh the tube Freeze the cell pellet liquid nitrogen 196 C Place samples in liquid nitrogen 30 C freezer or freezer 2 Perform protein extraction Add Extraction Labeling Buffer 20 ul buffer 1 mg cells Mix thoroughly by vortexing until mixture is homogeneous Incubate at room temperature 10 min Constant rotation mixing Centrifuge suspension 10 000 x g for 30 min at 4 C Transfer the supernatant to a clean tube Place tube on ice Discard the pellet Protocol No PT3648 1 www clontech com Clontech Laboratories Inc Version No PR123821 ATakara Bio Company 20 Antibody Microarrays 380 amp 500 User Manual Vil Protein Extraction amp Labeling Small Scale Protocol continued 3 Measure protein concentration amp dilute sample Measure protein concentration Use Pierce s BCA Protein Assay Reagent Kit Dilute sample with Extraction Labeling Buffer 1 1 mg protein ml Final volume must be 2200 ul Proceed immediately to Section C If final volume is below 200 ul STOP Do not proceed to the next step Repeat the extraction
15. 5 ml Stock Incubation Buffer in a clean plastic bottle or tube Set up Incubation Tray and label the four chambers Use a pen to mark exterior surface Slide 1 Incubation Slide 1 Wash Slide 2 Incubation Slide 2 Wash Add Incubation Buffer 5 ml To each chamber 2 Prepare protein sample mixes Label two 1 5 ml microfuge tubes Slide 1 Mix and Slide 2 Mix Slide 1 Mix Preparation Protein Sample A Cy5 100 ug Protein Sample B Cy3 100 ug Slide 2 Mix Preparation Protein Sample A Cy3 100 ug Protein Sample B Cy5 100 ug Leftover Samples A and B can be stored at 4 C short term storage or 20 C long term storage for later use in other applications e g Western blotting but stored protein samples are not recommended for future microarray analyses 3 Transfer protein sample mixes to Incubation Tray Transfer Incubation Buffer 5 ml To Slides 1 amp 2 Incubation chambers Add protein from Slide 1 Mix 20 ug To Slide 1 Incubation chamber Add protein from Slide 2 Mix 20 pg To Slide 2 Incubation chamber For samples derived from serum use 100 ug of protein since a large percentage 80 of the protein consists of immunoglobulin and albumin Incubate the tray at RT 10 min With gentle rocking Prepare Ab Microarray Slides Step 4 while incubating tray with protein sample mixes www clontech com Protocol No PT3648 1 Version No PR123821 27 Antibody Microarrays 380 amp 500 User Manual V
16. 6 the label may begin to interfere with antigen antibody binding 4 Sample calculation Sample A Cy5 Sample data BCA assay results protein concentration by BCA 0 18 mg ml 1 5 Absorbance results Aso Sample calculation Cy5 concentration in sample A so Esso x 10 1 5 250 000 x 10 B Protein concentration in sample BCA assay result average molecular weight x 10 0 18 60 000 x 10 B HM Dye protein Cy5 concentration in sample protein concentration in sample BM 3 um 2 For best results the dye protein ratio should be in the range of 2 4 When this ratio is significantly greater e g gt 6 the label may begin to interfere with antigen antibody binding Protocol No PT3648 1 www clontech com Clontech Laboratories Inc Version No PR123821 ATakara Bio Company 18 Antibody Microarrays 380 amp 500 User Manual VII Protein Extraction amp Labeling Small Scale Protocol PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING Follow the appropriate section below depending on your starting material crude tissue Section A or cultured cells Section B P A Extracting Protein from Crude Tissue MOUA 4 Chill the following items on ice or at 4 C e Extraction Labeling Buffer one small mortar amp pestle e two 1 5 ml microcentrifuge tubes 2 Perform protein extraction Frozen tissue 15 25 mg transfer to a prechilled mortar Add alumina 2 5 mg to the mortar Use t
17. Company Antibody Microarrays 380 amp 500 User Manual lil List of Components continued Ab Microarray 500 Slides Cat No 631790 2 Ab Microarray 500 slides 1 Storage Vial Protein Extraction amp Labeling Kit Cat No 631786 This kit is designed for first time users Use this kit to optimize the labeling reaction The goal is to obtain the correct dye molecule ratio to allow for reliable reproducible results between experiments 20 ml Extraction Labeling Buffer 20 ml 10X Desalting Buffer e 100 ul Blocking Buffer Visit our Antibody Arrays product page at www clontech com for a current list of products available for use with our Ab Arrays Clontech Laboratories Inc www clontech com Protocol No PT3648 1 ATakara Bio Company Version No PR123821 7 Antibody Microarrays 380 amp 500 User Manual IV Additional Materials amp Equipment Required The following materials are required but not supplied Alumina Sigma Cat No A 2039 for disintegrating tissue samples BCA Protein Assay Reagent Kit Pierce Biotechnology Cat No 23225 or 23227 Pierce s BCA Protein Assay Reagent Kit has been tested by our scientists and is approved for use with Ab Microarray procedures and reagents Bovine serum albumin BSA protein standard 0 1 M sodium carbonate buffer pH 8 3 Used in the preparation of body fluids for array analysis Cy5 mono Reactive Dye Pack GE Healthcare Cat No PA25001 Cy3 mon
18. Corporation ScanArray is a registered trademark of PerkinElmer Inc GenePix is a trademark of Axon Instruments BD Falcon is a trademark of Becton Dickinson and Company Clontech the Clontech logo and all other trademarks are the property of Clontech Laboratories Inc unless noted otherwise Clontech is a Takara Bio Company 2011 Clontech Laboratories Inc Clontech Laboratories Inc ATakara Bio Company www clontech com Protocol No PT3648 1 Version No PR123821 35
19. a Bio Company Version No PR123821 5 Antibody Microarrays 380 amp 500 User Manual lll List of Components Protocol No PT3648 1 Version No 6 PR123821 Store Ab Microarray Slides at 20 C Store all other components at 4 C Ab Microarray Express Buffer Kit Cat No 631795 2 Incubation Trays e 20 ml Extraction Labeling Buffer e 200 pl Blocking Buffer 20 ml 10X Desalting Buffer e 90 ml Stock Incubation Buffer 2 Bottles 10 ml Background Reducer e 20 ml Wash Buffer A e 20 ml Wash Buffer B e 20 ml Wash Buffer C e Antibody Microarrays 380 amp 500 User Manual PT3648 1 e Ab Microarray Analysis Workbook A Microsoft Excel 97 98 file used for array data analysis This workbook must be downloaded from the Bioin formatics or Online Tools page of our web site at www clontech com The Ab Microarray Express Buffer Kit provides reagents suitable for two Ab Microarray experiments four slides total as described in this User Manual Ab Array 380 Disease Profiling Array Cat No 631797 2 Ab Microarray 380 slides e 1 Storage Vial Ab Array 380 Disease Profiling Kit Cat No 631796 4 Ab Microarray 380 slides 2 Storage Vials e 1 Ab Microarray Express Buffer Kit Cat No 631795 Ab Microarray 500 Kit Cat No 631798 4 Ab Microarray 500 slides 2 Storage Vials s 1 Ab Microarray Express Buffer Kit Cat No 631795 www clontech com Clontech Laboratories Inc ATakara Bio
20. ations Ei Attention Clontech Laboratories Inc ATakara Bio Company A Handling Ab Microarray Slides e Wear laboratory gloves whenever handling Ab Microarrays Alternatively use tweezers to manipulate slides e Always hold slides at the end nearest the affixed data label Note This label includes identifying information for the array B Orienting Microarrays 1 2 3 4 abcdefghlabcdefghlabcdefghlabcdefgh ee oo ee Ab Microarray OTkwWN OTRwWN abcdefgh abcdefgh abcdefgh abcdefgh oe oo ee w DOnaRwWN DOnRwWN abcdefgh abcdefgh abcdefgh abcdefgh oe oo oe iv OTRwWN OTaWNS abcdefgh abcdefgh abcdefgh abcdefgh oo o onnon Figure 2 Layout of the Ab Microarray 500 The Ab Microarray 500 contains at least 500 distinct antibodies arrayed in a 32 x 32 grid on a 75 x 25 x 1 mm glass slide Panel A Each antibody is printed in duplicate Panel B Darker dots at the corners represent Cy3 Cy5 labeled bovine serum albumin BSA spots which serve as orientation markers The open circles correspond to unlabeled BSA spots which serve as negative controls The Ab Microarray 380 has a similar layout to the Ab Microarray 500 The difference is that rows 5 and 6 are not printed in the Ab Microarray 380 C Choosing Your Protocol In completing an Ab Microarray analysis you have the option of using either a Large Scale or Small Scale Protein Extraction amp Labeling protocol Th
21. cesccsscesscovaseessabeasoecesscaveccesecsscesscceuscbecaees sueeddeensiacaseesaecvacesaaeevecoeaactesteeavee 34 List of Figures Figure 1A The Ab Microarray procedure Steps 1 3 eseessssesssesescsensescecseseeeceesescseaesescaeeececaeeeeesesaveeeeseeaneeaty 4 Figure 1B The Ab Microarray procedure Steps 4 5 cccssssessecssesessesescscsesececsesesceeeaeseaeeceececaseesesesateeeenasseaeaeeanes 5 Figure 2 The Ab Microarray Analysis Workbook contains four worksheets csscsssesesessseeeteeeeeeeeeeaeseaeeeeees 32 Figure 3 The Import amp Analyses worksheet has three sections Import Analyses and Sorting 10 0 0 32 Figure 4 The Sorting section of the Import amp Analyses worksheet 0 cscsecssesesseceeeseseeetecesseseeeeatseaeeeeees 33 List of Tables Table I Comparison of protein extraction amp labeling methods 0 0 eeceeseseeeeeeseseeeeeeseseseeeeececneeesecesaeeeeeeeeaeeenty 9 Customer Service Ordering Technical Support tel 800 662 2566 toll free tel 800 662 2566 toll free fax 800 424 1350 toll free fax 650 424 1064 web www clontech com e mail tech clontech com e mail orders clontech com Protocol No PT3648 1 www clontech com Clontech Laboratories Inc Version No PR123821 ATakara Bio Company 2 Antibody Microarrays 380 amp 500 User Manual l Introduction Clontech Laboratories Inc ATakara Bio Company The Ab Microarray 500 enables you to detect a wide variety
22. col No PT3648 1 ATakara Bio Company Version No PR123821 15 Antibody Microarrays 380 amp 500 User Manual VI Protein Extraction amp Labeling Large Scale Protocol continued F Determining Protein Concentration We recommend the BCA Protein Assay Reagent Kit from Pierce Biotechnology It has Protocol been tested by our scientists and shown to be compatible with our buffers Using other BCA reagents or kits could lead to errors in protein estimation 1 Standard curve Use bovine serum albumin BSA as your protein standard Dilute BSA to 0 02 mg ml 0 05 mg ml 0 1 mg ml 0 2 mg ml 0 3 mg ml 0 4 mg ml 0 5 mg ml Use 1 X Desalting Buffer 0 mg ml As the blank Measure each sample in triplicate 2 Subtract contribution of dyes Cy3 and Cy5 absorb at 562 nm Prepare protein blank Substitute BCA reagent with 1X Desalting Buffer Add an aliquot of your labeled protein 3 Sample plate layout ENEA A Std 0 Std 0 02 Std 0 05 Std 0 1 Std 0 2 Std 0 3 Std 0 4 Std 0 5 Proteinblank Protein blank Protein blank Protein blank mg ml mg ml mg ml mg ml mg ml mg ml mg ml mg ml Sample A Cy3 Sample A Cy5 Sample B Cy3 Sample B Cy5 Std 0 Std 0 02 Std 0 05 Std 0 1 Std 0 2 Std 0 3 Std 0 4 Std 0 5 Protein blank Protein blank Protein blank Protein blank mg ml mg ml mg ml mg ml mg ml mg ml mg ml mg ml Sample A Cy3 Sample A Cy5 Sample B Cy3 Sample B Cy5 Std 0 Std 0 02 Std 0 05 Std 0 1 Std 0 2 Std 0 3 Std 0
23. e any residual traces of liquid Weigh the cell pellet Reweigh the tube Freeze the cell pellet liquid nitrogen 196 C Place samples in liquid nitrogen 30 C freezer or freezer 2 Perform protein extraction Add Extraction Labeling Buffer 20 ul buffer 1 mg cells Mix thoroughly by vortexing until mixture is homogeneous Incubate at room temperature 10 min Constant rotation mixing Centrifuge suspension 10 000 x g for 30 min at 4 C Transfer the supernatant to a clean tube Discard the pellet Clontech Laboratories Inc www clontech com Protocol No PT3648 1 ATakara Bio Company Version No PR123821 11 Antibody Microarrays 380 amp 500 User Manual VI Protein Extraction amp Labeling Large Scale Protocol continued 3 Measure protein concentration amp dilute sample Measure protein concentration Use Pierce s BCA Protein Assay Reagent Kit Dilute sample with Extraction Labeling Buffer 1 1 mg protein ml Final volume must be 21 mi Proceed immediately to Section D If final volume is below 1 mi STOP Do not proceed to the next step Repeat the extrac tion with a fresh sample or carry out small scale labeling starting at Section VII C P C Preparing Protein from Body Fluids Protocol Note A preliminary estimate of the protein concentration followed by a desalting procedure and a second protein assay is necessary to accurately measure the protein levels in body fluids Desalting may be performed at room temperat
24. e key differences between these two protocols are summarized in Table I TABLE I COMPARISON OF PROTEIN EXTRACTION amp LABELING METHODS Large Scale Tissues Cells amp Body Fluids Quantity of tissue or cells required for protein extraction Step 1 15 25 mg 50 200 mg Small Scale Quantities Needed for or Obtained from Specific Steps Tissues amp Cells Expected yield of total protein following protein extraction Step 1 250 500 ug 23mg Quantity needed for labeling Step 2 200 ug HECE Cosmea OO OOOO S O E gt The quantities and volumes given correspond to those needed for a single sample Recall that for each array analysis two different samples referred to as Samples A and B in Figure 1A are prepared and analyzed Both protocols yield sufficient protein to perform a single antibody microarray analysis The large scale method however yields more than enough protein for additional downstream analyses performed in conjunction with the antibody microarray procedure Note however that we do not recommend storing labeled protein for long periods because of the potential for protein degradation D Preventing Protein Degradation DO NOT USE protease inhibitors The Extraction Labeling Blocking Desalting Stock Incubation and Wash Buffers do not contain protease inhibitors because of their potential interference with protein labeling We obtain excellent results without protease inhibitors and thus do not recomm
25. e sampling method described above Steps 2 3 in which a portion of each protein sample is labeled with a portion of each fluorophore see Figure 1 By following our protocol you obtain an Internally Normalized Ratio INR for each antibody antigen pair on the microarray This sampling method con trols for differences in labeling efficiency After gel filtration Step 3 the four samples are combined in equal proportions to form two samples e Mix 1 which comprises A Cy5 and B Cy3 e Mix 2 which comprises A Cy3 and B Cy5 One microarray is incubated with Mix 1 the second microarray is incubated with Mix 2 In this setup Array 1 measures A Cy5 B Cy3 Ratio 1 while Array 2 measures B Cy5 A Cy3 Ratio 2 After the slides are scanned arrange the fluorescence data in our Ab Microarray Analysis Workbook Microsoft Excel 97 98 located on the the Bioinformatics or Online Tools page of our web site at www clontech com and obtain an Internally Normalized Ratio for each coordinate on the array by computing Ratio 1 Ratio 2 This value now represents the abundance of an antigen in Sample A relative to that of Sample B Please see Section B 2 for more details B Controls Several bovine serum albumin BSA spots are included on all Ab Microarrays Some of these spots prelabeled with Cy3 and Cy5 serve as positive controls and as dis cussed in Section V serve as orientation markers to help you identify the printed area of the micr
26. end their use during protein extraction labeling or array detection We do however recommend that once you start the extraction you work quickly and proceed diligently towards the array analysis step www clontech com Protocol No PT3648 1 Version No PR123821 9 Antibody Microarrays 380 amp 500 User Manual VI Protein Extraction amp Labeling Large Scale Protocol P Protocol Protocol No PT3648 1 Version No 10 PR123821 PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING Follow the appropriate section below depending on your starting material crude tissue Section A cultured cells Section B or body fluids Section C A Extracting Protein from Crude Tissue 1 Chill the following items on ice or at 4 C e Extraction Labeling Buffer e one mortar amp pestle e two 2 ml microcentrifuge tubes e one 15 ml conical centrifuge tube 2 Perform protein extraction Frozen tissue 100 200 mg transfer to a prechilled mortar Add alumina 0 25 0 5 g to the mortar Use the pestle to grind the tissue until a paste is formed Add prechilled Extraction Labeling Buffer 1 2 ml to the mortar Mix the buffer into the paste using the pestle Use a micropipette tip to scrape the paste that adheres to the pestle back into the mortar Transfer the extract to a prechilled 2 ml microcentrifuge tube Rinse pestle with Extraction Labeling Buffer 1 2 ml Hold the pestle over the mortar rinse the pestle Combine the rinse with
27. he pestle to grind the tissue until a paste is formed Add prechilled Extraction Labeling Buffer 100 200 ul to the mortar Mix the buffer into the paste using the pestle Use a micropipette tip to scrape the paste that adheres to the pestle back into the mortar Transfer the extract to a prechilled 1 5 ml microcentrifuge tube Rinse pestle with Extraction Labeling Buffer 100 200 ul Hold the pestle over the mortar rinse the pestle Combine the rinse with the original extract in a 1 5 ml tube Centrifuge suspension 10 000 x g for 30 min at 4 C Transfer the supernatant to a prechilled 1 5 ml microcentrifuge tube Do not disturb the pellet Mix lysate by gently inverting the tube Clontech Laboratories Inc www clontech com Protocol No PT3648 1 ATakara Bio Company Version No PR123821 19 Antibody Microarrays 380 amp 500 User Manual Vil Protein Extraction amp Labeling Small Scale Protocol continued 3 Measure protein concentration amp dilute sample Measure protein concentration Use Pierce s BCA Protein Assay Reagent Kit Dilute sample with Extraction Labeling Buffer 1 1 mg protein ml Final volume must be 2200 ul Proceed immediately to Section C If final volume is below 200 ul STOP Do not proceed to the next step Repeat the extraction with a fresh sample and use more starting material B Extracting Protein from Cultured Cells Protocol Note For adherent cells that are 90 confluent we find that one 10
28. ill Antibody Array Incubation Protocol continued Protocol No PT3648 1 Version No 28 PR123821 4 Prepare Ab Microarray Slides for incubation Important Use gloved hands or tweezers to hold and manipulate the slides Never touch the array end of the slide Instead always hold the slide at the end nearest the affixed label The Ab array slides are supplied in Storage Buffer inside a green capped Storage Vial An empty storage vial for drying the slides is also provided The Storage Buffer contains glycerol and should be disposed of in a properly labeled waste container Decant the Storage Buffer from Press your gloved finger against the green capped Storage Vial the top of vial to keep the slides from falling out Wash Ab Microarray Slides as follows Transfer the slides into the dry clean Storage Vial provided with the slides Add Stock Incubation Buffer 30 ml To Storage Vial Cap Storage Vial and slowly invert 10 times Decant Storage Buffer while using your gloved finger to keep the slides from falling out Add Stock Incubation Buffer 20 mi To Storage Vial Cap Storage Vial and slowly invert 10 times Stand the vial upright in a rack Record each slide s lot number Designate one slide as Slide 1 and the other slide as Slide 2 5 Incubate Ab Microarray Slides with protein sample mixes Important Place each slide array side up into the appropriate incubation chamber in the Incubation Tray see Step 1 The array is pr
29. inted on the side to which the label is affixed Transfer Slide 1 from the Storage Vial to the Slide 1 Incubation chamber Transfer Slide 2 from the Storage Vial to the Slide 2 Incubation chamber Incubate the tray at RT 40 min With gentle rocking Note Use a micropipette tip to pry up one end of each slide while you gently rock the Incubation Tray once or twice This helps exchange liquid on all sides of the slide www clontech com Clontech Laboratories Inc ATakara Bio Company Antibody Microarrays 380 amp 500 User Manual Vill Antibody Array Incubation Protocol continued 6 Wash Ab Microarray Slides amp prepare for scanning Important Use gloved hands to hold and manipulate the slides Do not touch the array surface of the slides Instead hold the slides by their edges Add Wash Buffer A 5 ml To each wash chamber Transfer Slide 1 from the Slide 1 Incubation chamber to the Slide 1 Wash chamber Transfer Slide 2 from the Slide 2 Incubation chamber to the Slide 2 Wash chamber Incubate at RT 5 min With gentle rocking Remove the buffer from the chambers Add Wash Buffer B 5 ml To each wash chamber Incubate at RT 5 min With gentle rocking Remove the buffer from the chambers Add Wash Buffer C 5 ml To each wash chamber Incubate at RT 5 min With gentle rocking Rinse the slides as follows Transfer each slide into a 50 ml conical tube filled with Milli Q grade water with the slide label facing downward then pour off the
30. mple A Cy3 Sample A Cy5 Sample B Cy3 Sample B Cy5 Std 0 Std 0 02 Std 0 05 Std 0 1 Std 0 2 Std 0 3 Std 0 4 Std 0 5 Protein blank Protein blank Protein blank Protein blank mg ml mg ml mg ml mg ml mg ml mg ml mg ml mg ml Sample A Cy3 Sample A Cy5 Sample B Cy3 Sample B Cy5 Cc Std 0 Std 0 02 Std 0 05 Std 0 1 Std 0 2 Std 0 3 Std 0 4 Std 0 5 Proteinblank Protein blank Protein blank Protein blank mg ml mg ml mg ml mg ml mg ml mg m mg ml mg ml Sample A Cy3 Sample A Cy5 Sample B Cy3 Sample B Cy5 Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample ACy3 ACy3 A Cy3 ACy5 ACy5 ACy5 BCy3 BCy3 BCy3 B Cy5 B Cy5 B Cy5 H Protocol No PT3648 1 www clontech com Clontech Laboratories Inc Version No PR123821 ATakara Bio Company 24 Antibody Microarrays 380 amp 500 User Manual Vil Protein Extraction amp Labeling Small Scale Protocol continued 4 Calculate protein concentration AOD Sample OD protein sample OD protein blank 562 562 standard OD blank 0 mg ml 562 AOD Std OD Use the AOD and your standard curve to estimate protein concentration Typical concentration of desalted samples 0 8 mg protein ml P F Estimating the Average Number of Coupled Dye Molecules Protocol 1 Measure absorbance using a spectrophotometer Dilute sample with 1X Desalting Buffer 1 10 dilution Prepare blank with 1X Desalting Buffer Measure Cy3 absorbance at 552 nm
31. n E P E Removing Unbound Dye using PD 10 Desalting Columns Protocol Note Dye removal using PD 10 desalting columns manufactured by GE Healthcare Cat No 17 0851 01 may be performed at room temperature if you work quickly or else at 4 C 1 Label microfuge tubes amp columns and prepare buffer Label four PD 10 Desalting Columns A Cy3 A Cy5 B Cy3 B Cy5 Label four 2 ml microcentrifuge tubes A Cy3 A Cy5 B Cy3 B Cy5 Prepare 1X Desalting Buffer 100 ml Dilute 10X Desalting Buffer with Milli Q H O in clean plastic bottle Adjust the pH to 7 4 Protocol No PT3648 1 www clontech com Clontech Laboratories Inc Version No PR123821 ATakara Bio Company 14 Antibody Microarrays 380 amp 500 User Manual VI Protein Extraction amp Labeling Large Scale Protocol continued 2 Perform desalting Add 1X Desalting Buffer 3 x 5 ml each To each PD 10 column equilibration step Load the Cy3 and Cy5 labeled 500 ul each Allow protein to pass into column protein samples Add 1X Desalting Buffer 2 mi each To each PD 10 column Allow buffer to pass into columns Elute each column by adding 2 mi each To each PD 10 column 1X Desalting Buffer Collect flowthrough in the prelabeled 2 ml microcentrifuge tubes Store tubes on ice 3 Measure protein concentration amp dilute sample Measure protein concentration Use Pierce s BCA Protein Assay Reagent Kit see Section F Clontech Laboratories Inc www clontech com Proto
32. n every 20 min Add Blocking Buffer 4 ul To each tube Wrap each tube in foil amp incubate on ice or at 4 C 30 min Mix by inversion every 10 min Proceed immediately to Section D P D Removing Unbound Dye using Protein Desalting Spin Columns Ptotocol Note Dye removal using Protein Desalting Spin Columns which are manufactured by Pierce Biotechnology Inc Cat No 89849 or 89862 may be performed at room temperature if you work quickly or else at 4 C 1 Label microfuge tubes amp columns and prepare buffer Label four Protein Desalting Spin Columns A Cy3 A Cy5 B Cy3 B Cy5 Label four 2 ml microcentrifuge tubes A Cy3 A Cy5 B Cy3 B Cy5 Prepare 1X Desalting Buffer 5 ml Dilute 10X Desalting Buffer with Milli Q H O in clean plastic bottle Adjust the pH to 7 4 Protocol No PT3648 1 www clontech com Clontech Laboratories Inc Version No PR123821 ATakara Bio Company 22 Antibody Microarrays 380 amp 500 User Manual Vil Protein Extraction amp Labeling Small Scale Protocol continued 2 Prepare desalting columns Spin each column to remove storage buffer 1 500 x g for 2 min Add 1X Desalting Buffer 2 x 400 ul 1 500 x g for 2 min to each column amp spin after each buffer addition Discard flowthrough after each spin Attach prelabeled microfuge tubes to the corresponding columns 3 Perform desalting Load the Cy3 amp Cy5 labeled 100 yl each protein samples Allow samples to pass into columns Cent
33. n researchers find recipe for proteins and chips Nature 402 719 720 Goffeau A Barrell B G Bussey H Davis R W Dujon B Feldmann H Galibert F Hoheisel J D Jacq C Johnston M Louis E J Mewes H W Murakami Y Philippsen P Tettelin H amp Oliver S G 1996 Life with 6000 genes Science 274 546 563 7 Gosmanov A R Umpierrez G E Carabel A H Cuervo R amp Thomason D B 9 March 2004 Impaired expression and insulin stimulated phosphorylation of Akt 2 in muscle of obese patients with atypical diabetes Am J Physiol Endocrinol Metab 287 1 E8 E15 Haab B B Dunham M J amp Brown P O 2001 Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions Genome Biol 2 2 research0004 1 0004 13 Hodgkin J Plasterk R H amp Waterston R H 1995 The nematode Caenorhabditis elegans and its genome Science 270 410 414 Lander E S et al 2001 Initial sequencing and analysis of the human genome Nature 409 860 921 Simpson R J amp Dorow D S 2001 Cancer proteomics from signaling networks to tumor markers Trends Biotechnol 19 suppl S40 S48 Song Y McDuffie E Sobocinski G States D amp Albassam M 2002 Profiling of human endothelial cells human aortic smooth muscle cells amp human mac rophages responses to lipopolysaccharide stimulation using protein microarrays Poster presented at the
34. nd A for all samples A s Sample A Cy3 absorbance 552 nm of Sample A Cy3 absorbance 552 nm of blank A s Sample B Cy3 absorbance 552 nm of Sample B Cy3 absorbance 552 nm of blank A Sample A Cy5 absorbance 650 nm of Sample A Cy5 absorbance 650 nm of blank A Sample B Cy5 absorbance 650 nm of Sample B Cy5 absorbance 650 nm of blank 2 General considerations Assume that the average molecular weight of protein is 60 000 Da Protein concentration use the values obtained from the BCA method Molar extension coefficient lt 552 of Cy3 150 000 M7 cm 650 of Cy5 250 000 M cm Clontech Laboratories Inc www clontech com Protocol No PT3648 1 ATakara Bio Company Version No PR123821 17 Antibody Microarrays 380 amp 500 User Manual VI Protein Extraction amp Labeling Large Scale Protocol continued 3 Sample calculation Sample A Cy3 Sample data BCA assay results protein concentration by BCA 0 18 mg ml Absorbance results A 0 9 s52 Sample calculation Cy3 concentration in sample A lt x 10 0 9 150 000 x 10 a Protein concentration in sample BCA assay result average molecular weight x 10 0 18 60 000 x 10 I HM Dye protein Cy3 concentration in sample protein concentration in sample BEMI sum 2 For best results the dye protein ratio should be in the range of 2 4 When this ratio is significantly greater e g gt
35. o Reactive Dye Pack GE Healthcare Cat No PA23001 Cy5 and Cy3 are fluorescent dyes that have distinct emission spectra 1 5 ml and 2 0 ml microcentrifuge tubes 15 ml and 50 ml conical centrifuge tubes e g BD Falcon conical centrifuge tubes Disposable PD 10 Desalting Columns GE Healthcare 17 0851 01 These columns are recommended for the Large Scale Protein Extraction amp Labeling Protocol e Protein Desalting Spin Columns Pierce Biotechnology Cat Nos 89849 or 89862 These columns are recommended for the Small Scale Protein Extraction amp Labeling Protocol Mortar amp pestle for grinding tissue Rocking platform to provide a constant see saw motion during slide incubation and washing Swinging bucket centrifuge with adaptors for spinning 50 ml tubes Microcentrifuge Spectrometer capable of measuring absorbance at 552 and 650 nm Microarray slide scanner You may use any scanner that is compatible with 75 x 25 x 1 mm slides and capable of dual color analysis The scanner must be capable of measuring Cy5 and Cy3 fluorescent labels Microsoft Excel 97 98 or later software application Used for calculating Internally Normalized Ratios based on fluorescence data from a microarray analysis Protocol No PT3648 1 www clontech com Clontech Laboratories Inc Version No PR123821 ATakara Bio Company 8 Antibody Microarrays 380 amp 500 User Manual V Important Pre Protocol Consider
36. oarray Other BSA spots not labeled with fluorophore serve as negative controls The coordinates of all BSA spots are given on the Certificate of Analysis Using the Ab Microarray Analysis Workbook to Calculate Internally Normalized Ratios The Ab Microarray Analysis Workbook is a Microsoft Excel 97 98 file that converts your fluorescence data into Internally Normalized Ratios INRs for each coordinate on the array As described in the Introduction Section I the INR calculated by our workbook is a numerical value that represents the abundance of antigen in Sample A relative to that of Sample B To get started Connect to the Bioinformatics or Online Tools page of our web site at www clontech com and download a copy of the workbook that corresponds to the Slide Lot Number of your Microarray The Microarray Slide Lot Number is given on the data label affixed to the glass slide NOTE The Microarray Slide Lot Number differs from that of the assembled Kit The Kit Lot Number is shown on the Certificate of Analysis and on the labels affixed to Boxes 1 and 2 www clontech com Protocol No PT3648 1 Version No PR123821 31 Antibody Microarrays 380 amp 500 User Manual IX Analysis of Results continued 2 Launch Microsoft Excel Then open the Microarray Analysis Workbook Upon opening the workbook you will notice that it contains four worksheets The names of these sheets appear on tabs at the bottom of the workbook window Figure
37. orm that is compatible with commercially available microarray scanners All arrayed antibodies are carefully tested for specificity and sensitivity Those that display a high degree of cross reactivity are eliminated from the final product For a complete list of the arrayed antibodies including Swiss Prot ID numbers of the target antigens please visit the Bioinformatics or Online Tools page of our web site at www clontech com Procedural Overview The Ab Microarray protocol outlined in Figures 1A amp 1B is a fluorescence based procedure in which solid phase antibody is used to capture fluorescently labeled antigen The entire procedure from sample preparation to array scan ning takes one day to complete Measuring protein abundances with Ab Microarrays consists of the following five main steps see Section II for details and troubleshooting information Step 1 Extract protein from cells or whole tissue Step 2 Label protein with Cy5 and Cy3 dyes Step 3 Remove unbound dye by gel filtration Step 4 Incubate labeled protein with Ab Microarrays Step 5 Scan microarrays to measure bound antigen www clontech com Protocol No PT3648 1 Version No PR123821 3 Antibody Microarrays 380 amp 500 User Manual ll Protocol Overview amp Troubleshooting Guide Harvesting amp Storage of Sample After collecting the pellet in a tube wash pellet 3X with PBS Remove the last wash Spin and remove the additional PBS Measure the pelle
38. rifuge to elute desalted protein 1 500 x g for 2 min Detach and cap microfuge tubes Store tubes on ice 4 Measure protein concentration amp dilute sample Measure protein concentration Use Pierce s BCA Protein Assay Reagent Kit see Section E Clontech Laboratories Inc www clontech com ATakara Bio Company Protocol No PT3648 1 Version No PR123821 23 Antibody Microarrays 380 amp 500 User Manual VII Protein Extraction amp Labeling Small Scale Protocol continued E Determining Protein Concentration We recommend the BCA Protein Assay Reagent Kit from Pierce Biotechnology It has Protocol been tested by our scientists and shown to be compatible with our buffers Using other BCA reagents or kits could lead to errors in protein estimation 1 Standard curve Use bovine serum albumin BSA as your protein standard Dilute BSA to 0 02 mg ml 0 05 mg ml 0 1 mg ml 0 2 mg ml 0 3 mg ml 0 4 mg ml 0 5 mg ml Use 1 X Desalting Buffer 0 mg ml As the blank Measure each sample in triplicate 2 Subtract contribution of dyes Cy3 and Cy5 absorb at 562 nm Prepare protein blank Substitute BCA reagent with 1X Desalting Buffer Add an aliquot of your labeled protein 3 Sample plate layout BSS ee Ee ae ee ea ae ee ee A Std 0 Std 0 02 Std 0 05 Std 0 1 Std 0 2 Std 0 3 Std 0 4 Std 0 5 Protein blank Protein blank Protein blank Protein blank mg ml mg ml mg ml mg ml mg ml mg m mg ml mg ml Sa
39. t size and record it on the tube or in a lab notebook Freeze the pellet immediately Protein Concentration If the protein concentration is less than 1 1 mg ml DO NOT proceed to the next step Cy3 Fluorescent Dye monfunctional NHS ester Extraction Labeling 110 pl Buffer q E Trial Labeling Before proceeding with the array experiment perform a labeling Sample A 470 yl Sample Preparation Sample B Cell pellet or crude tissue i a Freeze thaw b Add Extraction Labeling Buffer c Incubate for 10 min d Centrifuge e Collect supernatant Complex solution of total cellular protein a Measure protein concentration b Dilute each sample to 1 1 mg ml c Split and combine with dye 470 ul 470 ul 470 yl Cy5 Fluorescent Dye monfunctional NHS ester 110 H e reaction on a test sample A Cy3 A Cy5 B Cy3 B Cy5 Use any available cell line 30 ul If the substitution degree 30 pl f is higher than 4 dilute 30 ul fresh Cy dyes with a 30 ul larger volume of the a Incubate at 4 C for 90 min Extraction Labeling Buffer b Add Blocking Buffer c Incubate at 4 C for 30 min You must obtain a d Remove unbound dye with desalting columns substitution degree between 1 and 4 in order to proceed y Large Scale Labeling shown Final labeled protein concentration l should be 0 2 mg ml using PD 10 y R columns for desalting g z
40. ttings are too high the slides should be rescanned using lower power settings If you are using a scanner other than the GenePix 4000B instrument adjust the laser power and PMT if possible to obtain a signal within the range of 2 500 9 000 FU for the control spots Some scanners such as the PerkinElmer ScanArray instrument adjust the laser settings based on a pre scan of the slide these types of scanners generally calculate the correct settings so adjustments are not normally required However it is still useful to check the signal intensities of the control spots as the automatic detection settings based on the pre scan may be incorrect Protocol No PT3648 1 www clontech com Clontech Laboratories Inc Version No PR123821 ATakara Bio Company 30 Antibody Microarrays 380 amp 500 User Manual IX Analysis of Results T Attention Clontech Laboratories Inc ATakara Bio Company A General Tips for Microarray Data Analysis In order to use our Ab Microarray Analysis Workbook as described below in Part B you must first calculate the Cy5 Cy3 fluorescent signal ratios for all coordinates on each array This calculation can usually be done with your array analysis software e g GenePix Pro The Cy5 Cy3 values are required to calculate Internally Normalized Ratios INRs as described below in Part B Antibody Array Data Analysis Features e A Internally normalized results Internal normalization refers to th
41. ube contains sufficient dye to label 1 mg of total protein You will need at least 1 ml of protein at a concentration of 1 1 mg ml to proceed with the fluorescent labeling protocol 1 Label tubes amp prepare dye solutions Label four 1 5 ml microcentrifuge tubes A Cy3 A Cy5 B Cy3 B Cy5 Add Extraction Labeling Buffer 110 pl To Cy3 dye tube Vortex dye solution 20 sec Centrifuge dye solution 10 sec moderate speed Add Extraction Labeling Buffer 110 ul To Cy5 dye tube Vortex dye solution 20 sec Centrifuge dye solution 10 sec moderate speed 2 Mix dyes with protein samples Add Cy3 solution 30 ul To tubes A Cy3 and B Cy3 Add Cy5 solution 30 ul To tubes A Cy5 and B Cy5 Add Protein Sample A 470 ul To tubes A Cy3 and A Cy5 Add Protein Sample B 470 ul To tubes B Cy3 and B Cy5 Invert each tube 3 times to mix the contents Centrifuge protein and dye mixture 10 sec moderate speed Clontech Laboratories Inc www clontech com Protocol No PT3648 1 ATakara Bio Company Version No PR123821 13 Antibody Microarrays 380 amp 500 User Manual VI Protein Extraction amp Labeling Large Scale Protocol continued 3 Incubate dye sample mixtures Wrap each tube in foil amp incubate on ice or at 4 C 90 min Mix by inversion every 20 min or incubate on a rocker at 4 C Add Blocking Buffer 4 ul To each tube Wrap each tube in foil amp incubate on ice or at 4 C 30 min Mix by inversion every 10 min Proceed immediately to Sectio
42. ues in column J of the Import amp Analyses worksheet of the Ab Microarray Workbook The average INR should then be multiplied by 1 3 to obtain the upper threshold value and 0 77 to obtain the lower threshold value for that experi ment This practice should be done for every set of slides For example if a given experiment generated an average INR of 1 1 the threshold values would then be 1 43 1 1 X 1 3 and 0 85 1 1 X 0 77 Thus for this hypothetical experiment a valid change in protein abundance would be any INR value gt 1 43 or lt 0 85 To validate your results you may wish to repeat the assay using individual anti bodies with a Western blot procedure www clontech com Protocol No PT3648 1 Version No PR123821 33 Antibody Microarrays 380 amp 500 User Manual X References Abbott A 1999a A post genomic challenge learning to read patterns of protein synthesis Nature 402 715 720 Abbott A 1999b How to spot a protein in a crowd Nature 402 715 717 Anderson K Potter A Baban D amp Davies K E 2003 Protein expression changes in spinal muscular atrophy revealed with a novel antibody array technology Brain 126 2052 2064 Blattner F R et al 1997 The complete genome sequence of Escherichia coli K 12 Science 277 1453 1470 Bult C J et al 1996 Complete genome sequence of the methanogenic archaeon Methanococcus jannaschii Science 273 1058 1073 Dalton R amp Abbott A 1999 Ca
43. ure if you work quickly or else at 4 C 1 Measure protein concentration amp dilute sample Measure protein concentration Use Pierce s BCA Protein Assay Reagent Kit Dilute sample with 0 1 M sodium carbonate buffer pH 8 3 4 mg protein ml 2 Perform desalting Add 0 1 M sodium carbonate buffer pH 8 3 3x5 mi To a PD 10 column equilibration step Load 2 5 ml Sample onto column Elute with 3 5 ml 0 1 M sodium carbonate buffer pH 8 3 Collect flowthrough in a clean 15 ml conical centrifuge tube 3 Measure protein concentration amp dilute sample Measure protein concentration Use Pierce s BCA Protein Assay Reagent Kit Dilute sample with Extraction Labeling Buffer 1 1 mg protein ml Final volume must be 21 mi Proceed immediately to Section D If final volume is below 1 mi STOP Do not proceed to the next step Repeat the extrac tion with a fresh sample or carry out small scale labeling starting at Section VII C Protocol No PT3648 1 www clontech com Clontech Laboratories Inc Version No PR123821 ATakara Bio Company 12 Antibody Microarrays 380 amp 500 User Manual VI Protein Extraction amp Labeling Large Scale Protocol continued P D Labeling Protein with Fluorescent Dye IMPORTANT Prepare dye solutions mix dyes with protein samples and centrifuge mixtures Protocol rapidly all without interruption After the Cy3 and Cy5 dyes are dissolved in buffer they must be used immediately Each dye t
44. with a fresh sample and use more starting material P C Labeling Protein with Fluorescent Dye Protocol IMPORTANT Prepare dye solutions mix dyes with protein samples and centrifuge mixtures rapidly all without interruption After the Cy3 and Cy5 dyes are dissolved in buffer they must be used immediately Each dye tube contains sufficient dye to label 1 mg of total protein 1 Label tubes amp prepare dye solutions Label four 1 5 ml microcentrifuge tubes A Cy3 A Cy5 B Cy3 B Cy5 Add Extraction Labeling Buffer 110 pl To Cy3 dye tube Vortex dye solution 20 sec Centrifuge dye solution 10 sec moderate speed Add Extraction Labeling Buffer 110 ul To Cy5 dye tube Vortex dye solution 20 sec Centrifuge dye solution 10 sec moderate speed 2 Mix dyes with protein samples Add Cy3 solution 6 ul To tubes A Cy3 and B Cy3 Add Cy5 solution 6 pl To tubes A Cy5 and B Cy5 Clontech Laboratories Inc www clontech com Protocol No PT3648 1 ATakara Bio Company Version No PR123821 21 Antibody Microarrays 380 amp 500 User Manual Vil Protein Extraction amp Labeling Small Scale Protocol continued Add Protein Sample A 94 ul To tubes A Cy3 and A Cy5 Add Protein Sample B 94 ul To tubes B Cy3 and B Cy5 Carefully pipette up and down several times to mix the contents Centrifuge protein and dye mixture 10 sec moderate speed 3 Incubate dye sample mixtures Wrap each tube in foil amp incubate on ice or at 4 C 90 min Mix by inversio
45. y more than 30 INR values are usually considered invalid if they are based on Cy5 Cy3 ratios in which one or more of the antigen signals is are less than twice the background signal Clontech Laboratories Inc ATakara Bio Company In theory an INR gt 1indicates that an antigen is more abundant in Sample A than in Sample B Conversely an INR lt 1 indicates that an antigen is less abundant in Sample A than in Sample B In reality experimental and assay variabilities must be taken into account when analyzing your results The two slide approach addresses the majority of variability that could be intro duced Although internal normalization improves the quality of your data and addresses potential differences in dye labeling between samples it is still not advisable to accept any INR value less than or greater than 1 0 as being a valid change In our experience INR values that are 21 3 or lt 0 77 indicate valid chang es that signify differences in protein abundance These threshold values were used successfully by Anderson et al 2003 It is important to note that the threshold values of 1 3 and 0 77 are based on an ideal average INR of 1 0 However an average INR of 1 0 is not normally obtained in actual experiments Thus threshold values should be calculated for each set of slides To calculate the threshold values it is necessary to calculate the aver age INR for the experiment The average INR can be obtained by averaging the val
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