Manual - RayBiotech, Inc.
Contents
1. Each package contains 2 or 4 membranes For up to 3 months unless stated otherwise or until expiration date IV ADDITIONAL MATERIALS REQUIRED Pipettors pipet tips and other common lab consumables e Orbital shaker or oscillating rocker Tissue paper blotting paper or chromatography paper e Adhesive tape or plastic wrap e Distilled or de ionized water 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 CONSIDERATIONS 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 Serum free or low serum containing media 0 2 FBS FCS is recommended If serum containing media is required testing an uncultured media sample as a negative control is ideal as many types of sera contain cytokines growth factors and other proteins e It is strongly recommended to add a protease inhibitor cocktail to cell and tissue lysate samples e Avoid using EDTA as an anti coagulant for collecting plasma if testing MMPs or other metal binding protei2. High background signals or all spots visible 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 Increase dilution of the sample or load less protein Exposed Too Long 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 pur
3. 6 oa 1 13 L15 IL 17 ai ige 1L 17F IL 20 L21 TAC JAMA KC Leptin aa Pro TNF 7 OPN OPG PRL RANTES SCF sTNFRI sTNFRII TACI TARC TPO TRANCE TROY MMP 9 alpha Pro TNF 8 OPN OPG PRL RANTES SCF sTNFRI STNFRII TACI TARC TPO TRANCE TROY MMP 9 alpha o P Q R s T U V w Xx Y z AA A E Eotaxin g E Fas Feg 1 CD36 CTLA 4 CXCL16 Decorin Dkk 1 Cadherin EGF Eotaxin1 2 Epigen Selectin Ligand RIIB E Eotaxin E Fas Feg 2 CD36 CTLA 4 CXCL16 Decorin Dkk 1 Gian EGF Eotaxin1 2 Epigen odin gand RIIB IL 1 IL 1 IL 2R IL 12 3 aoka De IL 1ra IL 2 sue IL 3 IL 4 IL 5 IL 6 IL 9 IL 10 IL 11 p40 IL 1 IL 1 IL 2R IL 12 4 IL 1 IL 2 IL 3 IL 4 IL 5 IL 6 IL 9 IL 10 IL 11 alpha beta 5 alpha p40 5 ga Lungkine MadCAM 1 MCP 1 MDC MFG MIG MIP MI MIP 2 lls MIP 3 MMP 2 Selectin E8 alpha gamma alpha beta L MFG MIP 1 MIP 1 MIP 3 MIP 3 6 cclectin Lungkine MadCAM 1 MCP 1 MDC aa MIG we ae MIP 2 ane ee MMP 2 7 a VCAM 1 VEGF ca a Si BLANK BLANK BLANK BLANK BLANK BLANK POS 8 E VCAM 1 VEGF a Ti Fi BLANK BLANK BLANK BLANK BLANK BLANK POS POS Positive Control Spot NEG Negative Control Spot BLANK Blank Spot NOTE Protein alternative names www raybiotech com via the Resources Page accession numbers 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 membra
4. 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 30 minutes at RT 4 Aspirate blocking buffer from each well with a pipette B Sample Incubation 5 Pipette 2 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 washes 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 2 ml of the prepared Biotinylated Antibody Cocktail into each well and incu
5. signal intensities is 5 10 comparing favorably with ELISA testing CV 10 15 ll HOW IT WORKS Here s how it works Array support YYYYY pi Samples lt Incubation of aa with arrayed antibody supports 12 hrs Cocktail of Cockta OO Incubation with Labeled e o 0 4 streptavidin ive Incubation with labeled Streptavidin 1hrs signals l Data analysis E and graph VA Z ea lil COMPONENTS AND STORAGE Store kit at lt 20 C immediately upon arrival Kit must used within the 6 month expiration date STORAGE TEM COMPONENT AAM CYT 6 2 AAM CYT 6 4 AAM CYT 6 8 TEMPERATURE AFTER THAWING 1 Antibody Arrays 2 membranes 4membranes 8 membranes 20 C 2 Blocking Buffer 1 vial 25 ml 2vials 25 ml ea 4vials 25 ml ea 3 Biotinylated Antibody Cocktail 2 vial Avials 8 vials eae 1 000X HRP Streptavidin 2 vials Concentrate i Eva My 50 ul ea 5 20X Wash Buffer Concentrate 1 vial 10 ml 1 vial 20 ml 2 vial 20 ml i 6 20X Wash Buffer II Concentrate 1vial 10m 1vial 20ml 2 vial 20 ml anh 7 2X Cell Lysis Buffer Concentrate 1 vial 10 ml 1 vial 16 ml 2 vial 16 ml 8 Detection Buffer C 1 vial 1 5 ml 1vial 2 5 ml 2 vial 2 5 ml 9 Detection Buffer D 1 vial 1 5 ml 1vial 2 5 ml 2 vial 2 5 ml 10 4 Well Incubation Tray w Lid 1 tray 2 trays Room Temperature Other Kit Components Plastic Sheets Array Map Template User Manual
6. RayBio C Series Mouse Cytokine Antibody Array C6 For the semi quantitative detection of 97 mouse proteins in serum plasma cell culture media and other liquid samples types Patent Pending Technology User Manual Revised March 30 2015 Cat AAM CYT 6 2 2 Sample Kit Cat AAM CYT 6 4 4 Sample Kit Cat AAM CYT 6 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 Storage Additional Materials Required Sample Tips and General Considerations A Sample Collection P reparation and Storage B Sample Types and Recommended Dilutions Amounts C Handling Membranes D Incubations and Washes Chemiluminescence Detection Tips 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 l Storage Typical Results A Control Spots B Data Extraction C Data Analysis Array Map Troubleshooting Guide Interpreting the Results n ubation OW UO WU WOWAWWOAN A HDHUUUNN HP PWN PPRPPPrPPB PBPWPRPPPPROO O I INTRODUCTION New te
7. bate 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 membranes 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
8. chase price Kodak X Omat is a registered trademark of Eastman Kodak Company Microsoft Excel is a registered trademark of Microsoft Corporation This product is for research use only N Re 2010 RayBiotech Inc 15
9. chniques such as cDNA microarrays have enabled us to analyze global gene expression However almost all cell functions are executed by proteins which cannot be studied simply through DNA and RNA techniques Experimental analysis clearly shows disparity can exist between the relative expression levels of mRNA and their corresponding proteins Therefore analysis of the proteomic profile is critical The conventional approach to analyzing multiple protein expression levels has been to use 2 D SDS PAGE coupled with mass spectrometry However these methods are slow expensive labor intensive and require specialized equipment Thus effective study of multiple protein expression levels can be complicated costly and time consuming Moreover these traditional methods of proteomics are not sensitive enough to detect most cytokines typically at pg ml concentrations Cytokines broadly defined as secreted cell cell signaling proteins distinct from classic hormones or neurotransmitters play important roles in inflammation innate immunity apoptosis angiogenesis cell growth and differentiation They are involved in most disease processes including cancer obesity and inflammatory and cardiac diseases Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool to study cell signaling pathways Regulation of cellular processes by cytokines is a complex dynamic process often involving multiple proteins Positive an
10. d negative feedback loops pleiotrophic effects and redundant functions spatial and temporal expression of or synergistic interactions between multiple cytokines even regulation via release of soluble forms of membrane bound receptors all are common mechanisms modulating the effects of cytokine signaling As such unraveling the role of individual cytokines in physiologic or pathologic processes generally requires consideration and detection of multiple cytokines rather than of a single cytokine RayBio C Series Antibody Arrays have several advantages over detection of cytokines using single target ELISA kits 1 More Data Same or Less Sample Antibody 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
11. ed Antibody Cocktail Incubation O HRP Streptavidin Incubation NOTE 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 i NoP ti N A 2 Blocking Buffer TSEAN Pipet
12. ee 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 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 a
13. embrane is marked by a dash or number in the upper left corner e 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 e 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 O Sample Incubation O Biotinylat
14. ere 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 XI ARRAY MAP A B c D E F G I J K L M N 1 POS POS NEG NEG 6Ckine ALK 1 AREG BLC CT 1 CD27 ee CD30 Da Ligand Ligand 2 POS POS NEG NEG 6Ckine ALK 1 AREG BLC CT 1 CD27 ox CD30 as Ligand Ligand 3 FES Fractakne SP case ace am STR SME ral HGF ii IGFBP IGFBP IGF 2 Ligand 1 Ligand gamma 5 6 a E ce e e l e l am e Ee a e a ae A E y a l a Ligand 1 Ligand gamma 5 6 5 E 11 13 L15 IL 17 ae IL 17E IL 17F IL 20 21 TAC JAMA KC Leptin si
15. 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 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 Array
16. nes longer No signals not even the positive controls spots Degradation of components due to improper storage Store entire kit at lt 20 C Do not use kit after expiration date See storage guidelines Improper preparation or dilution of the HRP Streptavidin 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
17. ns e Avoid using hemolyzed serum or plasma as this may interfere with protein detection and or cause a higher than normal background response 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 General tips for preparing serum plasma cell culture media urine and 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 if necessary Normalize samples by loading equal amounts or equal dilutions e Cell Cultured Media Neat no dilution needed e Serum amp Plasma 2 fold to 10 fold dilution e Other Body Fluids and Liquids Neat or 2 fold to 5 fold dilution e Cell and Tissue Lysates load 50 to 500 ug of total protein after a 5 fold to 10 fold dilution to minimize the effect of any detergent s Therefore the original lysate concentration should be 1 to 5 mg ml C Handling Membranes e The antibody printed side of each m
18. rea 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 multiple 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 Wh
19. s Sample 1 Sample 2 Control ees p 2 e ee o z ee r a A te b E e E E c E E B e 8 amp e ee 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 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 s
20. te 2 ml of Blocking Buffer into 3 Biotinylated Antibody Cocktail each vial Mix gently with a N A pipette 10 ul of 1 000X trate 9990 1 000X HRP Streptavidin Dilute 1 000 fold with Blocking Peon AA ee ee 4 h A ul of Blocking Buffer 10 ml of 1X Concentrate Buffer Mix gently with a pipette i working solution 5 20X Wash Buffer Concentrate Dilute each 20 fold with distilled or deionized 10mlof20X concentrate 190mlofwater 6 20X Wash Buffer II Concentrate water 200mlof1X working solution 7 2X Cell Lysis Buffer Concentrate Dilute 2 fold with distilled or deionized 10 mlof 2X concentrate 10mlofweter water 20mlof1X working solution 8 Detection Buffer C 9 Detection Buffer D No Preparation N A 10 8 Well Incubation Tray w Lid 1 vial is enough to test 1 membrane 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 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
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