Antibody Microarray User Manual (Clontech)
Contents
1. 1 2 3 4 Measure the concentration of protein in your sample using the bicinchoninic acid BCA method Smith et al 1985 Note We recommend using Pierce s BCA Protein Assay Kit Dilute your sample to 4 mg protein ml by adding the appropriate volume of 0 1 M sodium carbonate buffer pH 8 3 Equilibrate a PD 10 Desalting Column with 15 ml of 0 1 M sodium carbonate buffer pH 8 3 Load 2 5 ml of your sample on the PD 10 column Protocol PT3648 1 www bdbiosciences com BD Biosciences Clontech Version PR23092 13 Antibody Microarrays User Manual V Antibody Array Detection Protocol continued 5 9 After the sample has passed into the column place a clean 15 ml conical centrifuge tube under the column to collect the flow through in Step 6 With aclean 15 ml tube in place to collect the flow through add 3 5 ml of 0 1 M sodium carbonate buffer pH 8 3 to the column Measure protein concentration using the bicinchoninic acid BCA method Smith et al 1985 Note We recommend using Pierce s BCA Protein Assay Kit Dilute each sample to 1 1 mg protein ml by adding the appropriate volume of Extraction Labeling Buffer In order to proceed with Part D the final volume must be 1 ml Proceed immediately with Part D D Labeling Protein with Fluorescent Dye Important Complete Steps 1 10 rapidly without interruption Once the Cy3 and Cy5 dyes are dissolved in buffer they must be used i2. software application Used for calculating Internally Normalized Ratios based on fluorescence data from a microarray analysis Internet connection To download the Ab Microarray Analysis Workbook a Microsoft Excel 97 98 file from www clontech com BD Biosciences Clontech www bdbiosciences com Protocol PT3648 1 10 Version PR23092 Antibody Microarrays User Manual IV General Considerations 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 unique identifying information for the array Orienting Microarrays e To assist you in identifying the different blocks of the printed area after scanning spots of Cy3 Cy5 labeled bovine serum albumin BSA have been printed on the slide These BSA spots serve as orientation markers that you can use to align the grid of your array analysis software The Cy3 Cy5 labeled spots are located at or near the outermost corners of the printed area The precise coordinates are given on the Product Analysis Certificate included with your microarrays e Note that the data label is affixed to the printed surface of the slide e Please see the enclosed Product Analysis Certificate for the identity and location of all array spots Using the Ab Microarray Storage Chamber e Ab Microarrays are supplied inside a green
3. Analysis of Results continued a OO Figure 3 The third worksheet Import amp Analyses contains formulas that perform arithmetic operations on the fluorescence 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 A Cy5 relative fluorescence units where Ratio 1 B Cy3 relative fluorescence units B Cy5 relative fluorescence units and Ratio 2 ____ 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 Click on the Import amp Analyses tab to make it the active the window 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 calcu lates Ratio 1 Ratio 2 and places these values in the next column which in Figure 3 is labeled R R IMPORT Paste Slide Paste Slide 3 Q 1 Cy5 Cy3 2 Cy8 Cy3 3 Step 1 Ratio Ratio amp z 23 gogl Ab Ag Slide 1 Slide 2 R R The Import amp Analyses worksheet has three sections Import Analyses
4. Mix 2 A Cy3 B Cy5 Array Detection Figure 1 The Ab Microarray procedure This figure continues on the next page BD Biosciences Clontech www bdbiosciences com 6 Protocol PT3648 1 Version PR23092 Antibody Microarrays User Manual l Introduction amp Protocol Overview continued Array Detection I g Mix 1 Mix 2 10 50 ug protein 10 50 ug protein 5 ml Incubation Buffer x oN y v 7 Incubation Tray Incubation Wash Incubation Wash l Slide 1 Chambers Slide 2 Chambers a Incubate at room temperature for 30 min b Place microarray slides array side up in the incubation chambers using forceps or gloved hands LE oe FA Incubation Tray Incubation Wash paar wa Slide 1 Chambers Slide 2 A y Incubate at room temperature for 30 min with constant rocking motion v Transfer slides to their respective wash chambers and begin washing y Dry slides by centrifugation v Scan Figure 1 The Ab Microarray Procedure continued Protocol PT3648 1 www bdbiosciences com Version PR23092 Step 4 Array incubation 2 hr Step 5 Scanning Microarrays 30 min BD Biosciences Clontech 7 Antibody Microarrays User Manual l Introduction amp Protocol Overview continued Internally normalized results By following our protocol you obtain an Internally Normalized Ratio INR for each antibody antigen pa
5. Transfer the extract to a pre chilled 2 ml microcentrifuge tube While holding the pestle over the mortar rinse the pestle with 1 2 ml of Extraction Labeling Buffer Combine the rinse with the original extract ina 2 ml tube Use a second 2 ml tube if the volume exceeds the tube s capacity Centrifuge the suspension at 10 000 x g for 30 min While taking care not to disturb the pellet transfer the supernatant to a pre chilled 15 ml conical centrifuge tube Gently invert the tube to mix the lysate Measure protein concentration using the bicinchoninic acid BCA method Smith et al 1985 Note We recommend using Pierce s BCA Protein Assay Kit Dilute each sample to 1 1 mg protein ml by adding the appropriate volume of Extraction Labeling Buffer In order to proceed with Part D the final volume must be 1 ml Proceed immediately with Part D BD Biosciences Clontech www bdbiosciences com Protocol PT3648 1 12 Version PR23092 Antibody Microarrays User Manual V Antibody Array Detection Protocol continued B Extracting Protein from Cells 1 00 10 11 Centrifuge 50 150 mg of cells in a pre weighed centrifuge tube Note 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 Decantthe su
6. 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 380 Workbook BD Biosciences Clontech www bdbiosciences com Protocol PT3648 1 20 Version PR23092 Antibody Microarrays User Manual VI Analysis of Results continued If your Antibody Microarray is one in which antibodies are printed in duplicate side by side spots the worksheet will also calculate an Average R R and an Average INR for each antibody antigen Ab Ag pair 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 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 6 389 Paste Ste 3 the data into columns H I p and J Then sort the data in columns H I and J by Average INR column J Ab Ag Average R R Average INR Figure 4 The Sorting section of the Import amp Analyses worksheet Note the view shown is that from the Ab Microarray 380 Workbook 8 Choose Data gt Sort and sort the data by Average INR in ascending or descending order Protocol PT3648 1 www bdbiosciences com BD Biosciences Clontech Version
7. capped Storage Vial Do not remove the microarray slides until you have labeled your protein samples and are ready to start the incubation Section V F An empty green capped Storage Vial is also supplied Use this vial to dry the microarray slides as described in Section V Protocol PT3648 1 www bdbiosciences com BD Biosciences Clontech Version PR23092 Antibody Microarrays User Manual V Antibody Array Detection Protocol PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING The Ab Microarray is designed to compare protein abundances among two biological tissue cell or body fluid samples In the following protocol we refer to these samples as Sample A and Sample B While preparing Samples A and B for array analysis you may find it helpful to refer to Figure 1 A Extracting Protein from Crude Tissue Before starting chill the following items on ice or at 4 C Oo AOON e 11 12 13 14 Extraction Labeling Buffer one mortar amp pestle two 2 ml microcentrifuge tube one 15 ml conical centrifuge tubes Transfer 100 200 mg of frozen tissue to a pre chilled mortar Add 0 25 0 5 g of alumina to the mortar Use the pestle to grind the tissue until a paste is formed Add 1 2 ml of pre chilled Extraction Labeling Buffer Mix the buffer into the paste using the pestle When you finish use a micropipette tip to scrape the paste that adheres to the pestle back into the mortar
8. 4 15 16 17 18 19 20 Record each slide s serial number and assign one slide to the Slide 1 Mix and one slide to the Slide 2 Mix Remove the slides one by one from the Storage Vial and place each array side up in the tray chamber containing the Incubation Buffer Slide Mix to which ithas been assigned The array is printed on the side to which the label is affixed Incubate the slides at room temperature for 30 min with gentle rocking Every 10 min perform the following manipulation to assist the exchange of liquid on all sides of the slide Use a micropipette tip to pry up one end of the slide while you gentle rock the Incubation Tray once or twice Add 5 ml of Incubation Buffer prepared in Step F 1 to each wash chamber Transfer the slides to their respective wash chambers Incubate at room temperature for 5 min with gentle rocking Remove the buffer from the wash chambers Add 5 ml of Wash Buffer 1 Incubate at room temperature for 5 min with gentle rocking Repeat Steps 15 17 using Wash Buffer 2 then using Wash Buffer 3 And so on until you have washed each Slide with each of the Wash Buffers 1 7 Dry the slides It is important to remove as much moisture as possible from the surface of the slides before the water evaporates passively We recommend the following method a Using gloved hands and holding the slides by their edges only place the slides array end up in the empty green capped Stora
9. CLONTECH Innovative Tools to Accelerate Discovery Antibody Microarrays User Manual PT3648 1 PR23092 Published 20 March 2002 See List of Components for storage conditions FOR RESEARCH USE ONLY Antibody Microarrays User Manual Table of Contents I Introduction amp Protocol Overview ll List of Components lll Additional Materials and Equipment Required IV General Considerations V Antibody Array Detection Protocol A B C D E F Extracting Protein from Crude Tissue Extracting Protein from Cells Preparing Protein from Body Fluids Labeling Protein with Fluorescent Dye Removing Unbound Dye Desalting Detecting Labeled Protein with the Ab Microarray VI Analysis of Results Vil References Vill Related Products List of Figures Figure 1 Figure 2 Figure 3 Figure 4 The Ab Microarray Procedure The Ab Microarray Analysis Workbook is composed of three worksheets The Import amp Analyses worksheet has three sections Import Analyses and Sorting The sorting section of the Import amp Analyses worksheet Notice to Purchaser This product is intended to be used for research purposes only It is not to be used for drug or diagnostic purposes nor is it intended for human use BD Biosciences Clontech products may not be resold modified for resale or used to manufacture commercial products without written approval of BD Biosciences Clontech Atlas and
10. Falcon are trademarks of Becton Dickinson and Company Milli Q is a registered trademark of Millipore Corporation Microsoft is a registered trademark of Microsoft Corporation GenePix is a trademark of Axon Instruments 2002 Becton Dickinson and Company BD Biosciences Clontech www bdbiosciences com 2 10 11 12 12 13 13 14 15 16 19 23 23 6 7 19 20 21 Protocol PT3648 1 Version PR23092 Antibody Microarrays User Manual l Introduction amp Protocol Overview With several genome sequencing projects nearing completion attention is turning towards understanding the complete complement of proteins the proteome One goal of proteomics is to measure changes in the expression levels of entire sets of proteins to gain a full view of biological processes and disease states To help accelerate this avenue of proteomics BD Biosciences Clontech has developed the Ab Microarray a powerful new chip based technology for profiling hundreds of proteins simultaneously The Ab Microarray is composed of hundreds of distinct monoclonal antibodies printed at high density on aglass microscope slide Using this innovative detection platform and our simple yet robust array protocol you can measure the abundances of hundreds of proteins with a single experiment Ab Microarrays can be used to measure protein levels in virtually any biological sample including cells whole tissue and body fluids This array analysi
11. 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 Smith P K Krohn R I Hermanson G T Mallia A K Gartner F H Provenzano M D Fujimoto E K Goeke N M Olson B J amp Klenk D C 1985 Measurement of protein using bicinchoninic acid Anal Biochem 150 76 85 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 Zhou H Roy S Schulman H amp Natan M J 2001 Solution and chip arrays in protein profiling Trends Biotechnol 19 suppl 834 839 Vill Related Products For the latest and most complete listing of all BD Biosciences Clontech products please visit www clontech com e Ab Microarray 380 K1 847 1 The Ab Microarray 380 recognizes 378 distinct intracellular proteins e Atlas Glass Microarrays many e Atlas Plastic Microarrays many e Atlas cDNA Expression Arrays membrane based many Protocol PT3648 1 www bdbiosciences com BD Biosci
12. PR23092 21 Antibody Microarrays User Manual VI Analysis of Results continued C 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 by 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 When an INR gt 1 an antigen is more abundant in Sample A than in Sample B Conversely when an INR lt 1 an antigen is less abundant in Sample A than in Sample B This is the strict meaning of an INR But as you analyze your INRs you should also consider the underlying causes of an antigen s apparent scarcity A given antigen may be less abundant in one cell or tissue type compared to another if posttrans lational modifications obscure or alter the required epitope or if rates of degradation or expression differ Any or all of these possibilities may affect the levels of a protein as measured by the Ab Microarray Although internal normalization improves the quality of your data only you can decide how large or small an INR must be for it to qualify as a valid indicator of protein abundance In our experience INR values that are 2 0 or lt 0 5 indicate with very high probability that there is a difference in protein abundance Changes of 1 5 and lower can be accepted but with less confidence To validate you
13. anual V Antibody Array Detection Protocol continued E Removing Unbound Dye Desalting Use Amersham Biosciences PD 10 Desalting Columns to remove unbound label as follows As long as you work quickly Steps 1 7 can be performed at room temperature Otherwise if you have access to a cold room we suggest you complete Steps 1 7 at 4 C 1 Set up and label four PD 10 Desalting Columns and four 2 ml microcentrifuge tubes A Cy3 A Cy5 B Cy3 and B Cy5 Note Use the microcentrifuge tubes at Step 6 below to collect the final flowthrough Prepare 100 ml of 1X Desalting Buffer by diluting 10X Desalting Buffer with the appropriate volume of Milli Q grade HO Store 1X Desalting Buffer in a clean plastic bottle Note Be sure that 1X Desalting Buffer has a pH 7 4 Adjust the pH if necessary using dilute HCI or NaOH Equilibrate each column with 15 ml of 1X Desalting Buffer Apply the Cy3 and Cy5 labeled protein samples 500 ul each to the corresponding columns Allow the protein sample to pass into the column Add 2 ml of 1X Desalting Buffer to each column Allow the buffer to pass into the column to push the protein sample further along Place the 2 ml microcentrifuge tubes under the corresponding columns Elute each protein sample by applying 2 ml of 1X Desalting Buffer to each column Collect the flowthrough Store the tubes on ice Measure protein concentration us
14. e resuspension whole tissue must first be homogenized using a mortar amp pestle as described in Section V Extraction Labeling Buffer contains non denaturing BD Biosciences Clontech www bdbiosciences com Protocol PT3648 1 4 Version PR23092 Antibody Microarrays User Manual l Introduction amp Protocol Overview continued detergents which emulsify membrane bound protein but do not interfere with antibody binding This extraction protocol recovers 295 of the cell s total protein content This result is based on side by side comparison with a commonly used SDS boiling method Step 2 protein labeling results in the covalent attachment of multiple 2 6 fluorophores to each protein molecule This step showcases one of the key time saving features of our protocol Because protein extraction and labeling take place in the same buffer no buffer exchange steps are necessary to generate labeled antigen This not only saves you time but prevents sample loss as well The second step also consists of some of the most critical hands on manipula tions in the microarray protocol The manipulations summarized in Figure 1 Step 2 are necessary to control for variations in labeling efficiency To prevent the labeling step from skewing the array results Samples A and B are each split into two equal portions Each portion is then labeled with either Cy5 or Cy3 to produce four samples A Cy3 A Cy5 B Cy3 and B Cy5 Notice as well tha
15. e layout of your Ab Microarray please refer to the Product Analysis Certificate provided When available database accession numbers e g SWISS PROT and Locus Link of target antigens are also listed so that you can retrieve detailed information about the proteins profiled with your array You can also view the list of arrayed antibodies and review pertinent array specifica tions by visiting www clontech com Be sure to note the Lot Number of the Protocol PT3648 1 www bdbiosciences com BD Biosciences Clontech Version PR23092 3 Antibody Microarrays User Manual l Introduction amp Protocol Overview continued microarray you wish to review as the composition of each lot may vary slightly depending on antibody availability at the time of printing The arrayed antibodies can be purchased individually from BD Biosciences Pharmingen or BD Biosciences Transduction Labs If you need specific informa tion about one or more antibodies on the microarray please visit www bdbiosciences com and navigate to the appropriate technical support pages Sensitivity and species cross reactivity of the Ab Microarray Because no two antibodies have exactly the same binding affinity K the lower detection limit of the Ab Microarray must be defined for each antibody But on average we find that the Ab Microarray can detect as little as 20 pg ml of a given target protein Although we have not thoroughly tested each microarray for its cross speci
16. ences Clontech Version PR23092 23
17. es reactivity it is likely that many of the epitopes recognized by arrayed antibodies are conserved among closely or even distantly related species In practice however it is difficult to predict how a given microarray will perform for different species We recommend you review the array specific antibody list available at www clontech com to decide which Ab Microarray is suitable for your experi mental system Overview of the Ab Microarray Procedure The Ab Microarray Protocol outlined in Figure 1 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 scanning takes just one day to complete Note that Ab Microarrays do not measure absolute concentrations Instead they provide a relative measure of protein abundance that is the protein levels in one sample Sample A are compared to those of a second sample Sample B Measuring protein abundances with Ab Microarrays consists of five main steps 1 Extracting protein 2 Labeling protein with Cy5 and Cy3 dyes 3 Removing unbound dye 4 Incubating labeled protein with Ab Microarrays 5 Scanning microarrays to measure bound antigen In Step 1 total protein is extracted from 50 200 mg of cells or whole tissue The samples are first centrifuged to produce a pellet Then after a single freeze thaw cycle each pellet is resuspended in Extraction Labeling Buffer Befor
18. ge Vial provided Important Do not touch the array surface b Cap the vial and centrifuge the slides at 1 000 x g for 25 min at room temperature c 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 Important Do not touch the array surface when removing the slides Instead hold the slides by their edges Scan the slides with a microarray scanner 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 Note Slides should be scanned lt 24 hours after drying BD Biosciences Clontech www bdbiosciences com Protocol PT3648 1 18 Version PR23092 Antibody Microarrays User Manual VI Analysis of Results 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 B Using the Ab Microarray Analysis Workbook to Calculate Internally Normalized Ratios The Ab Microarray Analysis Workboo
19. ing the BCA method See notes below Notes for determining protein concentration We recommend using Pierce s BCA Protein Assay Kit e Use bovine serum albumin BSA as your protein standard Construct a standard curve using BSA solutions that are 0 02 0 05 0 1 0 2 0 3 0 4 and 0 5 mg ml We recommend you measure each standard and protein sample in triplicate Use 1X Desalting Buffer as a blank i e as the 0 mg ml sample e Because Cy3 and Cy5 absorb at 562 nm you will need to subtract the dyes contribution to the overall ODsgo To do this prepare a protein blank that contains an aliquot of your labeled protein sample and 1X Desalting Buffer substituted for BCA reagent Calculate the AODs OD562 protein sample OD 562 protein blank Use the AODsggo and your standard curve to estimate protein concentration e Using the BCA method we typically find that desalted samples contain 0 2 mg protein ml Protocol PT3648 1 www bdbiosciences com BD Biosciences Clontech Version PR23092 15 Antibody Microarrays User Manual V Antibody Array Detection Protocol continued 10 Optional Estimate the average number of dye molecules covalently coupled to each protein Follow the protocol in the Amersham Product Specification Sheet but first see the notes below Notes for estimating the average number of coupled dye molecules e Unless you have more precise measurements for the distribution of protein mass i
20. ir on the microarray Internal normalization refers to the 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 This sampling method controls for differences in labeling efficiency For example if Cy5 reacts more efficiently with your protein targets than Cy3 does your results will be biased in favor of the sample labeled with Cy5 With our method however these differences are eliminated because each protein extract is allowed to react with each label thus generating four individual samples A Cy3 A Cy5 B Cy3 and B Cy5 After gel filtration these four samples are combined in equal proportions to form just two samples Mix 1 which comprises A Cy5 and B Cy3 and Mix 2 which comprises A Cy3 and B Cy5 These mixes are then incubated with the two Ab Microarrays One microarray is incubated with Mix 1 the second with Mix 2 In this set up Array 1 measures A Cy5 B Cy3 Ratio 1 Whereas Array 2 measures B Cy5 A Cy3 Ratio 2 After the slides are scanned and the fluorescence data are arranged in our Ab Microarray Analysis Workbook Microsoft Excel 97 98 an Internally Normalized Ratio is obtained for each coordinate on the array by computing Ratio 1 Ratio2 This value represents the abundance of an antigen in Sample A relative to that of Sample B Please see Section VI for more details Controls Several bovine serum albumin BSA s
21. k is a Microsoft Excel 97 98 file that helps you quickly convert 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 1 Connect to www clontech com and download a copy of the workbook that corresponds to the Lot Number of your Microarray The Microarray Lot Number is given on the data label affixed to the glass slide Note The Microarray Lot Number differs from that of the assembled Kit The Kit Lot Number is shown on the Product Analysis Certificate and on the labels affixed to Boxes 1 and 2 2 Launch Microsoft Excel Then open the Microarray Analysis Work book Upon opening the workbook you will notice that it contains three worksheets The names of these sheets appear on tabs at the bottom of the workbook window Figure 2 Array Ab List gt Import amp Analyses Figure 2 The Ab Microarray Analysis Workbook is composed of three worksheets The 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 Protocol PT3648 1 www bdbiosciences com BD Biosciences Clontech Version PR23092 19 Antibody Microarrays User Manual VI
22. many of the antigens are under represented in one or both of your samples you may wish to add the maximum amount i e 50 ug For those cell lines and tissue samples tested so far we find that 10 20 ug of protein is usually sufficient for detecting differences in antigen abundance 7 Incubate the tray at room temperature for 30 min with gentle rocking 8 Meanwhile prepare the Ab Microarrays by washing the slides two times as follows Notes Use gloved hands or tweezers to hold and manipulate the microarrays Never touch the array end of the slide Instead always hold the slide at the end nearest the affixed label a While pressing your gloved finger against the top of the vial to keep the slides from falling out decant the Storage Buffer from the green capped Storage Vial Note The Storage Buffer contains glycerol and should be disposed of in a properly labeled waste container b Add 30 ml of Stock Incubation Buffer Cap the Storage Vial Then slowly invert the vial 10 times d Decant the Stock Incubation Buffer while using your gloved finger to keep the slides from falling out e Add 20 ml of Incubation Buffer prepared at above at Step F 1 Repeat Step c g Stand the vial upright in a rack O hh Protocol PT3648 1 www bdbiosciences com BD Biosciences Clontech Version PR23092 17 Antibody Microarrays User Manual V Antibody Array Detection Protocol continued 9 10 11 12 13 1
23. mmediately 1 Label four 1 5 ml microcentrifuge tubes A Cy3 A Cy5 B Cy3 and B Cy5 2 Dissolve the Cy3 dye in 110 ul of Extraction Labeling Buffer by adding the buffer directly to the tube in which the dye is supplied Note Our studies show that each tube of dye contains a quantity of dye that will optimally label 1 mg of total protein 3 Mix thoroughly by vortexing for 20 sec 4 Centrifuge the tube at moderate speed for 10 sec to recover the liquid in the bottom of the tube 5 Immediately add 50 ul of Cy3 solution to tubes A Cy3 and B Cy3 6 Prepare a solution of Cy5 dye in the same manner by following Steps 2 4 7 Immediately add 50 ul of Cy5 solution to tubes A Cy5 and B Cy5 8 Add 450 ul of Protein Sample A to tubes A Cy3 and A Cy5 9 Add 450 ul of Protein Sample B to tubes B Cy3 and B Cy5 10 Invert each tube three times to mix the contents Then centrifuge each tube at moderate speed for 10 sec to recover the liquid in the bottom of the tube 11 Incubate all four tubes on ice or at 4 C for 90 min Mix each tube by inversion every 20 min 12 Add 4 ul of Blocking Buffer to each tube 13 Incubate each tube on ice or at 4 C for 30 min Mix each tube by inversion every 10 min 14 Proceed immediately with Part E BD Biosciences Clontech www bdbiosciences com Protocol PT3648 1 14 Version PR23092 Antibody Microarrays User M
24. n your sample assume that the average molecular weight of protein is 60 kDa Use the value determined with the BCA method not the Az method to calculate the molar concentration of protein in your sample e Measure Cy3 absorbance at 552 nm and Cy5 absorbance at 650 nm Then use the appropriate molar extinction coefficient e to determine the molarity of each 55 of Cy3 150 000 M cm of Cy5 250 000 M7 cm See the sample calculation below 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 11 Proceed immediately with Part F Sample calculation for Step E 10 If you use 1 cm cuvettes and you find that e Asso of Sample A Cy3 0 9 and e protein of Sample A Cy3 as measured by BCA assay 0 18 mg ml then a Cy3 in Sample A Cy3 0 9 150 000 x 10 6 uM b protein 0 18 g L 60 000 g mol x 10 3 uM c Cy3 protein 6 3 2 F Detecting Labeled Protein with the Ab Microarray 1 Prepare 45 ml of Incubation Buffer by mixing 4 5 ml of Background Reducer with 40 5 ml of Stock Incubation Buffer Store Incubation Buffer in a clean plastic bottle or tube We provide you with 80 ml of Stock Incubation Buffer You use 40 5 ml at this step to make Incubation Buffer and the remainder 39 5 ml at Step 8 b below 2 Setup the Incubation Tray pr
25. oninic acid BCA solution protein assay reagent Available from Pierce or prepare your own Smith et al 1985 Coligan et al 1995 e 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 e Cy5 mono Reactive Dye Pack Amersham Biosciences PA25001 e Cy3 mono Reactive Dye Pack Amersham Biosciences PA23001 Cy5 and Cy3 are fluorescent dyes that have distinct emission spectra Amersham Pharmacia supplies the Cy5 and Cy3 dyes as monofunctional N hydroxysuccinimide NHS esters in dried pre measured amounts The NHS ester is a functional group that reacts with primary amines The reaction produces a covalent bond which links the dye to the protein To use these dyes with the Ab Microarray follow the labeling procedure given in Section V 1 5 ml and 2 0 ml microcentrifuge tubes e 15 ml and 50 ml conical centrifuge tubes e g BD Falcon e Disposable PD 10 Desalting Columns Amersham Biosciences 17 0851 01 Mortar amp pestle for grinding tissue e Rocking platform To provide a constant see saw motion during slide incubation and washing e Swinging bucket centrifuge with adaptors for spinning 50 ml tubes e Microcentrifuge Microarray slide scanner You may use any scanner that is compatible with 75 x 25 x 1 mm slides and with dual color analysis using Cy5 and Cy3 fluorescent labels Microsoft Excel 97 98
26. ovided Note that it contains four separate chambers for incubating and washing microarrays 1 and 2 You may find it helpful to mark the exterior surface of the tray with a pen to remind you of these assignments Slide 1 Incubation Slide 1 Wash Slide 2 Incubation Slide 2 Wash BD Biosciences Clontech www bdbiosciences com Protocol PT3648 1 16 Version PR23092 Antibody Microarrays User Manual V Antibody Array Detection Protocol continued 3 Add 5 ml of Incubation Buffer to each incubation chamber of the Incubation Tray 4 Set up two 1 5 ml microcentrifuge tubes Label the tubes Slide 1 Mix and Slide 2 Mix 5 In the 1 5 ml microcentrifuge tubes combine Protein Samples A and B as follows e Slide 1 Mix combine 100 ug of Protein Sample A Cy5 with 100 ug of Protein Sample B Cy3 e Slide 2 Mix combine 100 ug of Protein Sample A Cy3 with 100 ug of Protein Sample B Cy5 Note If desired the remainders of 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 We do not recommend you use stored protein samples for future microarray analyses 6 Transfer 10 50 ug of protein from the Slide 1 Mix to the Slide 1 incubation chamber Transfer an equal quantity of protein from the Slide 2 Mix to the Slide 2 Incubation chamber Note The amount of protein you add is partly dependent on the source of your sample If you suspect that some or
27. pernatant and aspirate any residual traces of liquid Then reweigh the tube to determine the weight of the cell pellet Freeze your samples by placing them in liquid nitrogen 196 C or in a 80 C freezer Place your samples at room temperature and add Extraction Labeling Buffer to each Note Add 20 ul of Extraction Labeling Buffer for each mg of cells or tissue e g if your sample comprises 50 mg of cells or tissue add 1 ml of Extraction Labeling Buffer Mix thoroughly by vortexing Check to be sure the mixture is homogeneous Incubate the samples at room temperature for 10 min with constant rotation Centrifuge the suspension at 10 000 x g for 30 min at 4 C Transfer the supernatant to a clean tube Discard the pellet Measure protein concentration using the bicinchoninic acid BCA method Smith et al 1985 Note We recommend using Pierce s BCA Protein Assay Kit Dilute each sample to 1 1 mg protein ml by adding the appropriate volume of Extraction Labeling Buffer In order to proceed with Part D the final volume must be 1 ml Proceed immediately with Part D C Preparing Protein from Body Fluids To measure protein levels in body fluids you will need to complete the following buffer exchange desalting steps As long as you work quickly Steps 1 6 can be completed at room temperature Otherwise if you have access to a cold room we suggest you complete Steps 1 6 at 4 C
28. pots are included on all Ab Microarrays Some of these spots are pre labeled with Cy3 and Cy5 and as discussed in Section IV serve as orientation markers to help you identify the printed area of the microarray Other BSA spots not labeled with fluorophore serve as negative controls The coordinates of all BSA spots are given on the Product Analysis Certificate BD Biosciences Clontech www bdbiosciences com Protocol PT3648 1 8 Version PR23092 Antibody Microarrays User Manual ll List of Components Store Ab Microarrays at 20 C Store all other reagents at 4 C 2 Ab Microarrays Supplied in Storage Buffer inside a green capped Storage Vial 1 Storage Vial empty green capped 1 Incubation Tray e 10 mi Extraction Labeling Buffer e 100 ul Blocking Buffer e 20 mi 10X Desalting Buffer e 80 mi Stock Incubation Buffer 5 mi Background Reducer e 10 ml Wash Buffer 1 e 10 ml Wash Buffer 2 e 10 ml Wash Buffer 3 e 10 ml Wash Buffer 4 e 10 ml Wash Buffer 5 e 10 ml Wash Buffer 6 e 10 ml Wash Buffer 7 Ab Microarray Analysis Workbook A Microsoft Excel 97 98 file used for array data analysis This workbook must be downloaded from our website at www clontech com Version PR23092 9 Antibody Microarrays User Manual lll Additional Materials and Equipment Required The following materials are required but not supplied Alumina Sigma A 2039 for disintegrating tissue samples e Bicinch
29. r results you may wish to repeat the assay using individual antibodies with a Western blot procedure BD Biosciences Clontech www bdbiosciences com Protocol PT3648 1 22 Version PR23092 Antibody Microarrays User Manual Vil References Abbott A 1999 A post genomic challenge learning to read patterns of protein synthesis Nature 402 715 720 Abbott A 1999 How to spot a protein in a crowd Nature 402 715 717 Blattner F R et al 1997 The complete genome sequence of Escherichia coliK 12 Science 277 1453 1470 Bult C J etal 1996 Complete genome sequence of the methanogenic archaeon Methanococcus jannaschii Science 273 1058 1073 Coligan J E Dunn B M Ploegh H L Speicher D W amp Wingfield P T 1995 et seq Current Protocols in Protein Science John Wiley amp Sons Inc NY Dalton R amp Abbott A 1999 Can 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 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
30. s for example are specially formulated to minimize background binding Using Wash Buffers 1 7 in sequence as described in Section V D reduces the average background fluorescence to a level that may be up to 100 times lower than that of some target signals Protocol PT3648 1 www bdbiosciences com BD Biosciences Clontech Version PR23092 5 Antibody Microarrays User Manual Introduction amp Protocol Overview continued Sample Preparation 7 Sample A Sample B Cell pellet or crude tissue a Freeze thaw b Add Extraction Labeling al c Centrifuge d Collect supernatant Complex solution of total cellular protein a Measure protein concentration PL b Dilute each sample to 1 1 mg ml Cy3 Fluorescent Dye c Split and combine with dye Cy5 Fluorescent Dye monfunctional NHS ester monfunctional NHS ester Extraction Labeling 110 pl 450 ul 450 ul 450 ul 450 pl 110 ul Extraction Labeling Buffer y 4 Buffer i C U NK A Cy3 A Cy5 B Cy3 B Cy5 50 ul 50 yl t 50 pl 50 ul a Incubate at 4 C for 90 min b Add Blocking Buffer c Incubate at 4 C for 30 min d Filter through PD 10 Desalting Columns T Y s u 4 A Cy5 B Cy3 100 ug 100 ug Mix 1 A Cy5 B Cy3 y A B Cy5 100 ug protein 100 ug protein Step1 Protein extraction 1 2 hr Step 2 Protein labeling 2 3 hr Step 3 Removing unbound dye 1hr i
31. s system includes all necessary buffers for protein extraction labeling and detection an incubation tray and two identical Ab Microarrays Production of Ab Microarrays The production of our Ab Microarrays consists of two main steps First the surface of a glass microscope slide is chemically modified to present functional groups for covalent binding with antibodies Second antibodies are printed on the slide using a high precision robot Our proprietary chemistry ensures that even after immobilization antibodies remain active Because the Ab Microarray is immobilized on a glass slide only small volumes of sample and reagents are required to complete the analysis And because you detect captured antigens by fluorescence data collection is especially convenient slides can be scanned with commercially available microarray scanners Specifications of the Ab Microarray Ab Microarrays detect a wide variety of cytosolic and membrane bound proteins which together represent a broad range of biochemical pathways Our Extrac tion Labeling and Incubation Buffers each a unique blend of salts and deter gents ensure that even membrane bound receptors can be extracted from whole tissue or cells labeled with fluorophore and profiled with the appropriate microarray All antibodies on our Ab Microarrays have been extensively tested to verify their specificities Each was raised against a known protein To find out which protein and to review th
32. t in labeling proteins each of the dye solutions must also be split into equal portions so that both samples react with identical dye stocks After protein labeling unbound dye is removed in Step 3 by gel exclusion chromatography gel filtration This step which takes just a few minutes to complete using small disposable desalting columns removes excess dye and transfers the labeled protein into the Desalting Buffer ready for microarray analysis In Step 4 labeled protein is incubated with the Ab Microarrays The labeled samples are first combined to produce a mixture of Cy5 and Cy3 labeled proteins A Cy5 is combined with B Cy3 Mix 1 while A Cy3 is combined with B Cy5 Mix 2 Once combined and thoroughly mixed the fluorescently labeled antigens are ready for incubation with the Ab Microarray An aliquot comprising 10 50 ug of total protein from each mix is incubated with a separate array in separate chambers of the Incubation Tray see Figure 1 Following the 30 min incubation the microarrays are washed dried and scanned Step 5 Specially formulated wash buffers ensure optimal signal to noise ratios In developing the Ab Microarray we have paid special attention to the compo sitions of the Extraction Labeling Incubation and Wash Buffers By careful testing we have formulated unique combinations of salts detergents and polymers to produce buffers that will yield the highest possible signal to noise ratio Our Wash Buffer
Download Pdf Manuals
Related Search