Manual - RayBiotech, Inc.
Contents
1. Do not allow membranes to dry out during the experiment or they may become fragile and break OR high and or uneven background may occur Grasp membranes by the corners or edges only using forceps DO NOT touch printed antibody spots Incubations and Washes Perform ALL incubation and wash steps under gentle rotation or rocking motion 0 5 to 1 cycle sec using an orbital shaker or oscillating rocker to ensure complete and even reagent sample coverage Rocking rotating too vigorously may cause foaming or bubbles to appear on the membrane surface which should be avoided All washes and incubations should be performed in the Incubation Tray ITEM 10 provided in the kit Cover the Incubation Tray with the lid provided during all incubation steps to avoid evaporation and outside debris contamination Ensure the membranes are completely covered with sufficient sample or reagent volume during each incubation Avoid forceful pipetting directly onto the membrane instead gently pipette samples and reagents into a corner of each well Aspirate samples and reagents completely after each step by suctioning off excess liquid with a pipette Tilting the tray so the liquid moves to a corner and then pipetting is an effective method Optional overnight incubations may be performed for the following steps to increase overall spot signal intensities fo Sample Incubation 0 Biotinylated Antibody Cocktail Incubation fo HRP Streptavidin Incubation NOT2. INTERPRETING THE RESULTS A Control Spots Positive Control Spots POS controlled amount of biotinylated antibody printed onto the array Used for normalization and to orientate the arrays Negative Control Spots NEG buffer printed no antibodies used to measure the baseline responses Used for determining the level of non specific binding of the samples Blank Spots BLANK nothing is printed here Used to measure the background response B Data Extraction Visual comparison of array images may be sufficient to see differences in relative protein expression However most researchers will want to perform numerical comparisons of the signal intensities or more precisely signal densities using 2 D densitometry Gel Blot documentation systems and other chemiluminescent or phosphorescent detection systems are usually sold as a package with compatible densitometry software Any densitometry software should be sufficient to obtain spot signal densities from your scanned images One such software program ImageJ is available for free from the NIH website along with an array plug in We suggest using the following guidelines when extracting densitometry data from our array images e For each array membrane identify a single exposure that the exhibits a high signal to noise ratio strong spot signals and low background response Strong Positive Control Spot signals but not too strong that that they are bleeding into one
3. K L 1 POS1 POS1 POS2 POS2 POS3 POS3 ABL1 ABL1 ACK1 ACK1 ALK ALK 2 NEG NEG NEG NEG Axl Axl Blk Blk BMX BMX Btk Btk 3 Csk Csk Dtk Dtk EGFR EGFR EphA1 EphA1 EphA2 EphA2 EphA3 EphA3 4 EphA4 EphA4 EphAS EphAS EphA6 EphA6 EphA7 EphA7 EphA8 EphA8 EphB1 EphB1 5 EphB2 EphB2 EphB3 EphB3 EphB4 EphB4 EphB6 EphB6 ErbB2 ErbB2 ErbB3 ErbB3 6 ErbB4 ErbB4 FAK FAK FER FER FGFR1 FGFR1 FGFR2 FGFR2 iuias rame a isoform a isoform 7 Fer Fer FRK FRK Fyn Fyn Hck Hck HGFR HGFR IGF IR IGF IR 8 des as Itk Itk JAK1 JAK JAK2 JAK2 JAK3 JAK3 LCK LCK 9 LTK LTK Lyn Lyn MATK MATK M CSFR M CSFR MUSK MUSK NGFR NGFR 10 a aad eas oF PYK2 PYK2 RET RET ROR1 ROR1 ROR2 ROR2 11 ROS ROS RYK RYK SCFR SCFR SRMS SRMS_ SYK SYK Tec Tec 12 Tie 1 Tie 1 Tie 2 Tie 2 TNK1 TNK1 TRKB TRKB TXK TXK NEG NEG 13 Tyk2 Tyk2 TYRO10 TYRO10 VEGFR2 VEGFR2 VEGFR3 VEGFR3 ZAP70 ZAP70 POS4 POS4 POS Positive Control Spot NEG Negative Control Spot BLANK Blank Spot NOTE Protein alternative names accession numbers www raybiotech com via the Resources Page and official symbols can be accessed on 13 xII TROUBLESHOOTING GUIDE PROBLEM CAUSE RECOMMENDATION Chemiluminescent imager is not Contact image manufacturer working properly Too Short Exposure Expose the membranes longer No signals not even the positive
4. Too much HRP Streptavidin or Biotinylated Antibody Cocktail Prepare these signal enhancing components precisely as instructed Membranes dried out Do not let the membranes dry out during the experiment Cover the incubation tray with the lid to minimize evaporation Too High of Sample Protein Concentration Exposed Too Long Increase dilution of the sample or load less protein Decrease exposure time Insufficient Washing Ensure all the wash steps are carried out and the wash buffer is removed completely after each wash step Non specific binding Ensure the blocking buffer is stored and used properly 14 RayBio is the trademark of RayBiotech Inc The RayBio Cytokine Antibody Array C Series is patent pending technology developed by RayBiotech This product is intended for research only and is not to be used for clinical diagnosis Our produces may not be resold modified for resale or used to manufacture commercial products without written approval by RayBiotech Inc Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials Products are guaranteed for 6 months from the date of purchase when handled and stored properly In the event of any defect in quality or merchantability RayBiotech s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase price Kodak X Omat is a registered trademark of
5. controls spots Degradation of components due to improper storage Improper preparation or dilution of the HRP Streptavidin Store entire kit at lt 20 C Do not use kit after expiration date See storage guidelines Centrifuge vial briefly before use mix well and do not dilute more than 1000 fold Waiting too long before exposing The entire detection process should be completed in 10 15 minutes Positive controls spots signals visible but no other spots Low sample protein levels Decrease sample dilution concentrate samples or load more protein initially Skipped Sample Incubation Step Samples must be loaded after the blocking step Too Short of Incubations Ensure the incubations are performed for the appropriate time or try the optional overnight incubation s Uneven signals and or background Bubbles present on or below membrane Don t rock rotate the tray too vigorously or pipette the sample or reagent with excessive force Insufficient sample or reagent volume Load enough sample and reagent to completely cover the membrane Insufficient mixing of reagents Gently mix all reagents before loading onto the membrane especially the HRP Streptavidin and Biotin Antibody Cocktail Rocking Rotating on an uneven surface while incubating Rock rotate on a flat surface or the sample or reagent can pool to one side High background signals or all spots visible
6. E Overnight incubations should be performed at 4 C also with gentle rocking shaking Be aware that Vi longer incubations can also increase the background response so complete liquid removal and washing is critical CHEMILUMINESCENCE DETECTION TIPS Beginning with adding the detection buffers and ending with exposing the membranes should take no more than 10 15 minutes as the chemiluminescent signals may start to fade at this point Trying multiple exposure times is recommended to obtain optimum results A few seconds to a few minutes is the recommended exposure time range with 30 seconds to 1 minute being suitable for most samples Don t have time or the equipment to image your membranes Let the experts at RayBiotech image and analyze your membranes Contact us for pricing details Vil COMPONENT PREPARATION NOTE Thaw all reagents to room temperature immediately before use If wash buffers contain visible crystals warm to room temperature and mix gently until dissolved NOTE The Biotinylated Antibody Cocktail ITEM 3 and the HRP Streptavidin Concentrate ITEM 4 vials should be briefly centrifuged 1000 g before opening to ensure maximum recovery and mixed well as precipitates may form during storage TEM COMPONENT PREPARATION EXAMPLE 1 Antibody Arrays No Preparati N A 2 Blocking Buffer o Preparation Pipette 2 ml of Blocking Buffer into 3 Biotinylated Antibody Cocktail each v
7. Eastman Kodak Company Microsoft Excel is a registered trademark of Microsoft Corporation 15 Note 16 This product is for research use only 2010 RayBiotech Inc 17
8. NS A Sample Collection Preparation and Storage NOTE Optimal methods will need to be determined by each experimenter empirically based on researched literature and knowledge of the samples e If not using fresh samples freeze samples as soon as possible after collection e Avoid multiple freeze thaw cycles If possible sub aliquot samples prior to initial storage e It is strongly recommended to add a protease inhibitor cocktail to cell and tissue lysate samples e Avoid sonication of 1 ml or less as this can quickly heat and denature proteins e Most samples will not need to be concentrated If concentration is required a spin column concentrator with a chilled centrifuge is recommended e Always centrifuge the samples hard after thawing 10 000 RPM for 2 5 minutes in order to remove any particulates that could interfere with detection e The Cell Lysate can be prepared as follows For attached cells remove supernatant from cell culture wash cells twice with cold 1X PBS for suspension cells pellet the cells by spinning down the cells at 1500 rpm for 10 min making sure to remove any remaining PBS before adding Lysis Buffer Solubilize the cells at 2x10 cells ml in 1X Lysis Buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail Set Il see preparation note shown on page 7 under Component Preparation Section Pipette up and down to resuspend cells and rock the lysates gently at 2 8 C for 30 minutes T
9. RayBio C Series Human RTK Phosphorylation Antibody Array C1 For the semi quantitative detection of 71 phosphorylated human receptor tyrosine kinases RTKs in cell and tissue lysates Patent Pending Technology User Manual Revised April 14 2015 Cat AAH PRTK 1 2 2 Sample Kit Cat AAH PRTK 1 4 4 Sample Kit Cat AAH PRTK 1 8 8 Sample Kit Please read manual carefully before starting experiment RayBiotech Inc Hip the protein array pioneer company Tel Toll Free 1 888 494 8555 or 1 770 729 2992 Fax 1 770 206 2393 Website www raybiotech com Email info raybiotech com Vi VII VIII xI XII C Series TABLE Antibody Arrays TABLE OF CONTENTS Introduction Components and Stora Additional Materials Required Sample Tips and General Considerations A Sample Collection P a ee ee re ee eer reparation and Storage B Sample Types and Recommended Dilutions Amounts C Handling Membranes D Incubations and Washes Component Preparatio Protocol A Blocking B Sample Incubation C First Wash D Biotinylated Antibody Cocktail Incubation E Second Wash F HRP Streptavidin Inc G Third Wash Storage Typical Results Interpreting the Results A Control Spots B Data Extraction C Data Analysis Array Map Troubleshooting Guide n ubation OW MO WO WOW WOWOAN DA UUM MN HP PWN PPRPPPrPPB BPWPRPPPPROO O l INTRODUCTION Protein phosphor
10. URE AFTER THAWING 1 Antibody Arrays 2 membranes 4membranes 8membranes lt 20 C 2 Blocking Buffer 1 vial 25 ml 2 vials 25 ml ea Biotinylated Anti Phosphotyrosine F 2 8 C 2 peng oe MA avae iii for up to 3 days after dilution 4 1 000X HRP Streptavidin 1 vial 50 pl Concentrate 5 20X Wash Buffer I Concentrate 1vial 30ml 2 vial 30 ml ea 6 20X Wash Buffer II Concentrate 1 vial 30ml 2 vial 30 ml ea aor 7 2X Cell Lysis Buffer Concentrate 1 vial 5 ml 1 vial 5 ml 8 Detection Buffer C 1vial 1 5 ml 1vial 2 5 ml 9 Detection Buffer D 1vial 1 5 ml 1vial 2 5 ml 10 8 Well Incubation Tray w Lid 1 tray Room Temperature 11 Protease Inhibitor Cocktail 1 vial 2 vials lt 20 C 12 Phosphatase Inhibitor Cocktail II 1 vial 2 vials Other Kit Components Plastic Sheets Array Map Template User Manual Each package contains 2 or 4 membranes For up to 3 months unless stated otherwise or until expiration date IV ADDITIONAL MATERIALS REQUIRED e Pipettors pipet tips and other common lab consumables e Orbital shaker or oscillating rocker Tissue paper blotting paper or chromatography paper Adhesive tape or plastic wrap e Distilled or de ionized water e A chemiluminescent blot documentation system o CCD Camera o X Ray Film and a suitable film processor o Gel documentation system o Or another chemiluminescent detection system capable of imaging a western blot V SAMPLE TIPS AND GENERAL CONSIDERATIO
11. another is ideal The exposure time does not need to be identical for each array but Positive Control signals on each array image should have similar intensities e Measure the density of each spot using a circle that is roughly the size of one of the largest spots Be sure to use the same extraction circle dimensions area size and shape for measuring the signal densities on every array for which you wish to compare the results e For each spot use the summed signal density across the entire circle ie total signal density per unit area C Data Analysis NOTE RayBiotech offers Microsoft Excel based Analysis Software Tools for each array kit for automatic analysis Please visit the website at www raybiotech com or contact us for ordering information Once the raw numerical densitometry data is extracted the background must be subtracted and the data normalized to the Positive Control signals to analyze Background Subtraction Select values which you believe best represent the background If the background is fairly even throughout the membrane the Negative Control Spots NEG and or Blank Spots BLANK should be similar and are accurate for this purpose 11 Positive Control Normalization The amount of biotinylated antibody printed for each Positive Control Spot is consistent from array to array As such the intensity of these Positive Control signals can be used to normalize signal responses for comparison of results across mult
12. chemiluminescence imaging system such as a CCD camera recommended and expose NOTE Optimal exposure times will vary so performing multiple exposure times is strongly recommended See Section VI for additional details Storage 22 To store without direct pressure gently sandwich the membranes between 2 plastic sheets if not already tape the sheets together or use plastic wrap to secure them and store at lt 20 C for future reference IX TYPICAL RESULTS Typical results obtained with RayBio C Series Antibody Arrays Sample 1 Sample 2 Control oe ee EAN Be ee i S E op x i se gt i E E E Ga gt eo B e 2 e e e e m e The preceding figures present typical images obtained with RayBio C Series Antibody Arrays These membranes were probed with conditioned media from two different cell lines Membranes were exposed with Kodak X Omat film at room temperature for 1 minute Note the strong signals of the Positive Control spots in the upper left and lower right corners See below for further details on the control spots The signal intensity for each antigen specific antibody spot is proportional to the relative concentration of the antigen in that sample Comparison of signal intensities for individual antigen specific antibody spots between and among array images can be used to determine relative differences in expression levels of each analyte sample to sample or group to group 10 X
13. es using fresh buffer and aspirating out the buffer completely each time 8 Wash Buffer II Wash Pipette 2 ml of 1X Wash Buffer II into each well and incubate for 5 minutes at RT Repeat this 1 more time for a total of 2 washes using fresh buffer and aspirating out the buffer completely each time D Biotinylated Antibody Cocktail Incubation NOTE The Biotinylated Antibody Cocktail ITEM 3 must be prepared before use See Section VII for details 9 Pipette 1 ml of the prepared Biotinylated Antibody Cocktail into each well and incubate for 1 5 to 2 hours at RT OR overnight at 4 C 10 Aspirate biotinylated antibody cocktail from each well E Second Wash 11 Wash membranes as directed in Steps 7 and 8 F HRP Streptavidin Incubation NOTE The 1 000X HRP Streptavidin Concentrate ITEM 4 must be diluted before use See Section VII for details 12 Pipette 2 ml of 1X HRP Streptavidin into each well and incubate for 2 hours at RT OR overnight at 4 C 13 Aspirate HRP Streptavidin from each well G Third Wash 14 Wash membranes as directed in Steps 7 and 8 H Chemiluminescence Detection NOTE Do not allow membranes to dry out during detection 15 Transfer the membranes printed side up onto a sheet of chromatography paper tissue paper or blotting paper lying on a flat surface such as a benchtop 16 Remove any excess wash buffer by blotting the membrane edges with another piece of paper 17 Transfer and place the me
14. ial Mix gently with a N A pipette 1 000X HRP Streptavidin Dilute 1 000 fold with Blocking TOHI OFL Ques concentrati E 20 4 ul of Blocking Buffer 10 ml of 1X Concentrate Buffer Mix gently with a pipette s working solution 5 20X Wash Buffer Concentrate Dilute each 20 fold with distilled or deionized 10 ml of 20X concentrate 190 ml of water 6 20X Wash Buffer II Concentrate water 200mlof1X working solution 3 EEE a y z 7 2X Celitysis Buffer Concentrate Dilute 2 fold with distilled or deionized 10 ml of 2X concentrate 1Omlotweter water 20 mlof1X working solution 8 Detection Buffer C 9 Detection Buffer D No Preparation N A 10 8 Well Incubation Tray w Lid Pipette 60 ul of 1X Cell Lysis Buffer into the vial to prepare 100X Protease Inhibitor Cocktail concentrate 11 Protease Inhibitor Cocktail Add 180 ul of 1X Lysis Buffer into the vial to prepare 25X Phosphatase Inhibitor Cocktail Set II Concentrate Dissolve the powder thoroughly by gentle mixing 12 Phosphatase Inhibitor Cocktail II 1 vial is enough to test 2 membranes Only for use for preparing cell or tissue lysates General tips for preparing lysates and other common sample types can be found on the online Resources Page Note Prior to preparing cell or tissue lysates Add 20 ul Protease Inhibitor Cocktail Concentrate 100X and 80 ul Phosphatase Inhibitor Cocktail Set II Concentrate 25X into 1 9 ml 1X Lysis B
15. iple arrays much like housekeeping genes and proteins are used to normalize results of PCR gels and Western Blots respectively To normalize array data one array is defined as Reference Array to which the other arrays are normalized to The choice of the Reference Array is arbitrary NOTE The RayBio Analysis Software Tools always designate Array 1 Sample 1 as the Reference Array Next the simple algorithm below can be used to calculate and determine the signal fold expression between like analytes X Ny X y P1 P y Where P1 mean signal density of Positive Control spots on reference array P y mean signal density of Positive Control spots on Array y X y mean signal density for spot X on Array for sample y Wu X Ny normalized signal intensity for spot X on Array y For example Let s determine the relative expression for IL 6 on two different arrays Arrays 1 and 2 Let s assume that the duplicate signals for the IL 6 spots on each array are identical or that the signal intensity used in the following calculation is the mean of the two duplicates spots Also assume the following P1 2500 P2 2700 IL 6 1 300 IL 6 2 455 Then IL 6 N2 455 2500 2700 421 30 The fold increase of IL 6 N2 vs IL 6 1 421 3 300 1 40 fold increase or a 40 increase in the signal intensity of IL 6 in Array 2 vs Array 1 12 Xl ARRAY MAP A B C D E F G H I J
16. mbranes printed side up onto a plastic sheet provided lying on a flat surface NOTE Multiple membranes can be placed next to each other and fit onto a single plastic sheet Use additional plastics sheets if necessary 18 Into a single clean tube pipette equal volumes 1 1 of Detection Buffer C ITEM 8 and Detection Buffer D ITEM 9 Mix well with a pipette EXAMPLE 250 ul of Detection Buffer C 250 ul of Detection Buffer D 500 ul enough for 1 membrane 19 Gently pipette 500 ul of the Detection Buffer mixture onto each membrane and incubate for 2 minutes at RT DO NOT ROCK OR SHAKE Immediately afterwards proceed to Step 20 NOTE Exposure should ideally start within 5 minutes after finishing Step 19 and completed within 10 15 minutes as chemiluminescence signals will fade over time If necessary the signals can usually be restored by repeating washing HRP Streptavidin and Detection Buffers incubations Steps 11 19 20 Place another plastic sheet on top of the membranes by starting at one end and gently rolling the flexible plastic sheet across the surface to the opposite end to smooth out any air bubbles The membranes should now be sandwiched between two plastic sheets NOTE Avoid sliding the top plastic sheet along the membranes printed surface If using X ray film do not use a top plastic sheet so that the membranes can be directly exposed to the film 21 Transfer the sandwiched membranes to the
17. ransfer extracts to microfuge tubes and centrifuge at 14 000 x g for 10 min It is recommended that sample protein concentrations be determined using a total protein assay For incubation with the Phosphorylation Antibody Array use at a protein concentration of 50 1000 ug ml for cell lysates Lysates should be used immediately or aliquot and stored at 70 C Thawed lysates should be kept on ice prior to use If you experience high background you may further dilute your samples If signals are too weak the cell lysates can be pretreated by immunoprecipitations before incubation with array membranes Immunoprecipitations can be done using anti phosphotyrosine and protein A General tips for preparing lysate samples can be viewed on the online Resources page of the website B Sample Types and Recommended Dilutions Amounts NOTE Optimal sample dilutions and amounts will need to be determined by each experimenter empirically but the below recommendations may be used as a starting point Blocking Buffer ITEM 2 should be used to dilute samples Normalize by loading equal amounts of protein per sample e Cell and Tissue Lysates load 50 to 1000 ug of total protein after at least a 5 fold dilution to minimize the effect of any detergent s Therefore the original lysate concentration should be 250 ug to 5 mg ml Handling Membranes The antibody printed side of each membrane is marked by a dash or number in the upper left corner
18. uffer immediately before use Mix well Vill PROTOCOL NOTE Prepare all reagents and samples immediately prior to use See Sections V and VII ALL incubations and washes must be performed under gentle rotation rocking 0 5 1 cycle sec 1 Remove the kit from storage and allow the components to equilibrate to room temperature RT 2 Carefully remove the Antibody Arrays ITEM 1 from the plastic packaging and place each membrane printed side up into a well of the Incubation Tray ITEM 10 One membrane per well NOTE The antibody printed side is marked by a dash or number in the upper left corner A Blocking 3 Pipette 2 ml of Blocking Buffer ITEM 2 into each well and incubate for 1 hour at RT 4 Aspirate blocking buffer from each well with a pipette B Sample Incubation 5 Pipette 1 ml of diluted or undiluted sample into each well and incubate for 1 5 to 5 hours at RT OR overnight at 4 C NOTE Longer incubations can help maximize the spot signal intensities However doing so can also increase the background response so complete liquid removal and washing is critical 6 Aspirate samples from each well with a pipette C First Wash NOTE The 20X Wash Buffer Concentrates and II ITEM 5 and 6 must be diluted 20 fold before use See Section VII for details 7 Wash Buffer Wash Pipette 2 ml of 1X Wash Buffer into each well and incubate for 5 minutes at RT Repeat this 2 more times for a total of 3 wash
19. y arrays provide high content screening using about the same sample volume as traditional ELISA 2 Global View of Cytokine Expression Antibody array screening improves the chances for discovering key factors disease mechanisms or biomarkers related to cytokine signaling 3 Similar sometimes better Sensitivity As little as 4 pg ml of MCP 1 can be detected using the C Series array format In contrast our similar MCP 1 ELISA assay has a sensitivity of 40 pg ml of MCP 1 4 Increased Range of Detection ELISA assays typically detect a concentration range of 100 to 1000 fold however RayBiotech arrays can detect IL 2 at concentrations of 25 to 250 000 pg ml a range of 10 000 fold 5 Better Precision As determined by densitometry the inter array Coefficient of Variation CV of spot signal intensities is 5 10 comparing favorably with ELISA testing CV 10 15 ll HOW IT WORKS ary Biotin Ab K K ki K t Labeled streptavidin KAX e00 a J KAK t 2o te Ey t tal E Samples Incubation of Sample with arrayed antibody supports l 2 hrs Incubation with Biotinylated Ab L 2 hrs Incubation with labeled Streptavidin l hrs Detection of signals Data analysis and graph lil COMPONENTS AND STORAGE Store kit at lt 20 C immediately upon arrival Kit must used within the 6 month expiration date STORAGE ITEM COMPONENT N E S ene TEMPERAT
20. ylation plays an unusually prominent role in cell signaling development and growth The RayBio Human RTK Phosphorylation Antibody Array C1 is a very rapid convenient and sensitive assay to simultaneous detect multiple protein phosphorylations and can be used to monitor activation or function of important biological pathways RayBiotech is committed to developing a series of phosphorylation antibody arrays Our first product in this series is RayBio Human RTK Phosphorylation Antibody Array C1 which is specifically designed for simultaneously identifying the relative levels of phosphorylation of 71 different human receptor tyrosine kinases RTKs in cell lysates By monitoring the changes in protein tyrosine phosphorylation in your experimental model system you can verify pathway activation in your cell lines without spending excess time and effort in performing an analysis of immunoprecipitation and or Western Blot By using RayBio Human RTK Phosphorylation Antibody Array C1 treated or untreated cell lysate is added into antibody array membranes The antibody array membranes are washed and biotinylated anti phosphotyrosine antibody is used to detect phosphorylated tyrosines on activated receptors After incubation with HRP streptavidin the signals are visualized by chemiluminescence RayBio C Series Antibody Arrays have several advantages over detection of cytokines using single target ELISA kits 1 More Data Same or Less Sample Antibod
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