Manual - RayBiotech, Inc.
Contents
1. 10 Block sub arravs bv adding 400 ul of Blocking Buffer Item F into each well of Assembled Glass Slide and incubating at RT for 30 min Ensure there are no bubbles on the arrav surfaces 11 Immediatelv prior to sample incubation spin biotin labeled samples for 5 min at 10 000 rpm to remove anv particulates or precipitants Dilute samples with Blocking Buffer Note Recommended dilution of the biotin labeled samples with Blocking Buffer prior to incubation is 2 10 fold for cell culture supernatants 20 fold for serum plasma or 30 fold cell tissue lysate Note Optimal sample dilution factor will depend on the abundance of target proteins If the background or antigen specific antibody signals are too strong the sample can be diluted further in subsequent experiments If the signal is too weak more concentrated samples can be used 12 Completely remove Blocking Buffer from each well Add 400 ul of diluted samples into appropriate wells Remove any bubbles on array surfaces Incubate arrays with gentle rocking or shaking for 2 hours at RT or overnight at 4 C RayBio L Series Rat Antibody Array L 90 Protocol Note Avoid the flow of sample into neighboring wells 13 Dilute 20X Wash Buffer Concentrate Item G 20 fold with de ionized or distilled water Decant the samples from each well and wash 3 times with 800 ul of 1X Wash Buffer at RT with gentle rocking or shaking for 5 min per wash 14 Obtain a clean container2. CINC 3 IL 2 Osteopontin SPP1 ai w Ss a o ore pa ra 2228 22 E SS ce CS E I E a om o a os Petes Pas cre o 50 IL 10 77 RELM beta IL 12 IL 23 p40 78 ps www Pepo ms fp 7 7 EG VEGF PKI Fas TNFRSF6 y Insulin Positive 2 TGF beta2 RavBio L Series Rat Antibodv Arrav L 90 Protocol 20 VI Interpretation of Results A Explanation of Controls Spots 1 2 Positive Control spots POS1 POS2 POS3 are standardized amounts of biotinvlated IgGs printed directly onto the array All other variables being equal the Positive Control intensities will be the same for each sub array This allows for normalization based upon the relative fluorescence signal responses to a known control much as housekeeping genes or proteins are used to normalize results in PCR or Western blots respectively Negative Control NEG spots contain a protein containing buffer used to dilute antibodies printed on the array Their signal intensities represent non specific binding of Biotin conjugated anti Cytokines and or the Cy3 Conjugated Streptavidin Negative control signal intensities are usually very close to background signals in each sub array B Typical results obtained with RavBio L Series Rat Obesitv Antibodv Arrav L 90 The following figure shows the RavBio L Series Rat Antibodv Arrav 90 probed with serum sample The images were captured using a Axon GenePix laser scanner
3. Blank Incubations and Washes Cover incubation chamber with a Plastic Adhesive Strip Item J to prevent evaporation during incubation or wash steps particularly those lasting 2 hours or longer During incubation and wash steps avoid foaming and be sure to remove all bubbles from the sub array surface Perform all incubation and wash steps under gentle rotation or rocking motion 70 5 to 1 cycle sec Wash steps in Wash Buffer II and all incubation steps may be performed overnight at 4 C Avoid cross contamination of samples to neighboring wells To remove Wash Buffers and other reagents from chamber wells you may invert the Glass Slide Assembly to decant and aspirate the remaining liquid Unlike most Cy3 fluors the HiLyte Plus 532 used in this kit is very stable at RT and resistant to photobleaching on the RavBio L Series Rat Antibody Array L 90 Protocol 8 hvbridized glass slides However please protect glass slides from directly strong light and temperatures above RT IV Protocol Assay Diagram 1 Cell culture supernatants 2 Serum or plasma or cell tissue lysates 1 Serum sample ti 1 Preparation of sample 2 Dialysis of sample 2 Dialysis of sample Ei 3 Determination of protein concentration 3 Biotinylation of sample 4 Biotinylation of sample 7 7 5 4 Dialysis of biotinylated 5 Dialysis of biotinylated U sample sample 5 proceed to microarray analysis 6 proceed
4. The strong signals in row 20 and the upper left and lower right corners of each RayBio L Series Rat Antibody Array L 90 Protocol 21 arrav are Positive Controls which can be used to identifv the orientation and help normalize the results between arravs RayBio L Series Rat Antibody Array 90 If scanned using optimal settings 3 distinct signal intensities will be seen POS1 gt POS2 gt POS3 If all of these signals are of similar intensity try increasing or decreasing laser power and or signal gain settings Also in the absence of an external standard curve for each protein detected there is no means of assessing absolute or relative concentrations of different proteins in the same sample using immunoassays If you wish to obtain quantitative data ie RayBio L Series Rat Antibody Array L 90 Protocol 22 concentrations of the various analvtes in vour samples trv using our Quantibody Arrays instead c Background Subtraction Once you have obtained fluorescence intensity data you should subtract the background and normalize to the Positive Control signals before proceeding to analysis Most laser fluorescence scanner software have an option to automatically measure the local background around each spot For best results we recommend comparing signal intensities representing the MEDIAN background signals minus local background If your resulting fluorescence signal intensity reports do not include these values
5. add 70 ul dialyzed serum and 120 ul Labeling Buffer to RavBio L Series Rat Antibody Array L 90 Protocol 11 keep same total volume 212 ul c For labeling cell or tissue lysates transfer 30 ug 15 ul of 2 mg ml cell or tissue Ivsates into a tube and add labeling buffer Item K for a total volume of 300 ul Then add 3 3 ul of 1X Labeling Reagent Solution 6 Incubate the reaction solution at room temperature with gentle rocking or shaking for 30 min Mix the reaction solution by gently tapping the tube every 5 min 7 Add 3 ul Stop Solution Item D into each reaction tube and immediately dialyze as directed in Steps 2 3 Note Biotinylated samples can be stored at 20 C or 80 C until you are ready to proceed with the assay C Drying of the Glass Slide 8 Remove the package containing the Assembled Glass Slide Item E from the freezer Place unopened package on the bench top for approx 15 min and allow the Assembled Glass Slide to equilibrate to room temperature RT 9 Open package and take the Assembled Glass Slide out of the sleeve Do not disassemble the Glass Slide from the chamber assembly Place glass slide assembly in laminar flow hood or similar clean environment for 1 2 hours at RT Note Protect the slide from dust or others contaminants RavBio L Series Rat Antibody Array L 90 Protocol 12 D Blocking and Incubations Note Glass slide should be completelv drv before adding Blocking Buffer to wells
6. e g a column labeled as MED532 B532 you may need to subtract the background manually or change the default settings on your scanner s data report menu D Normalization of Array Data To normalize signal intensity data one sub array is defined as reference to which the other arrays are normalized This choice is arbitrary For example in our Analysis Tool Software described below the array represented by data entered in the left most column each worksheet is the default reference array You can calculate the normalized values as follows X Ny X y P1 P y RayBio L Series Rat Antibody Array L 90 Protocol 23 Where P1 mean signal intensity of POS spots on reference array P y mean signal intensity of POS spots on Array y X y mean signal intensity for spot X on Array y X Ny normalized signal intensity for spot X on Array y The RavBio Analvsis Tool software is available for use with data obtained using RayBio Biotin Label based Antibody Arrays You can copy and paste your signal intensity data with and without background into the Analysis Tool and it will automatically normalize signal intensities to the Positive Controls To order the Analysis Tool please contact us at 1 770 729 2992 or info raybiotech com for more information E Threshold of significant difference in expression After subtracting background signals and normalization to Positive Controls comparison of sig
7. e g pipette tip box or slide staining jar place the Assembled Glass Slide into the container with enough volume of 1X Wash Buffer to completely cover the entire assembly and remove any bubbles in wells Wash 2 times at RT with gentle rocking or shaking for 10 min per wash 15 Dilute 20X Wash Buffer Il Concentrate Item H 20 fold with de ionized or distilled water Decant the Wash Buffer from each well place the Assembled Glass Slide into the container with enough volume of 1X Wash Buffer Il to completely cover the entire assembly and remove any bubbles in wells Wash 2 times at RT with gentle rocking or shaking for 5 min per wash 16 Prepare 1X Cy3 Conjugated Streptavidin a Briefly spin down tube containing the Cy3 Conjugated Streptavidin Item I immediately before use b Add 1000 ul of Blocking Buffer into the tube to prepare a concentrated Cy3 Conjugated Streptavidin stock RavBio L Series Rat Antibody Array L 90 Protocol 14 solution Pipette up and down to mix gentiv do not store the stock solution for later use c Add 200 ul of Cy3 Conjugated Streptavidin stock solution into a tube with 800 ul of Blocking Buffer Mix gentiv to prepare 1X Cv3 Conjugated Streptavidin 17 Carefully remove Assembled Glass Slide from container Remove all of Wash Buffer II from the wells Add 400 ul of 1X Cy3 Conjugated Streptavidin to each sub array Cover the incubation chamber with the plastic adhesive strips Note Avoid e
8. in end stag heart failure patients relationshp between MMP 10 and LV remodeling J Cell Mol Med 2011 15 4 773 782 9 Kuranda K Berthon C Lep tre F et al Expression of CD34 in hematopoietic cancer cell lines reflects tightly regulated stem progenitor like state J Cell Biochem 2011 112 5 1277 1285 10 Toh HC Wang W W Chia WK et al Clinical Benefit of Allogenic Melanoma Cell Lysate Pulsed Autologous Dendritic Cell Vaccine in MAGE Positive Colorectal Cancer Patients Clin Chem Res 2009 15 7726 7736 11 Zhen Hou Cytokine array analysis of peritoneal fluid between women with endometriosis of different stages and those withoutendometriosi Biomarkers 2009 14 8 604 618 12 Yao Liang Tang et al Hypoxic Preconditioning Enhances the Benefit of Cardiac Progenitor Cell Therapy for Treatment of Myocardial Infarction by Inducing CXCR4 Circ Res 2009 109 197723 RayBio L Series Rat Antibody Array L 90 Protocol 27 RayBio L series Antibody Arrays are 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 six months from the date of shipment when handled and s
9. into a tube with 1 ml 1X Cell Lysis Buffer 2X Cell Lysis Buffer should be diluted 2 fold with deionized or distilled water e Homogenize the tissue according to homogenizer manufacturer instructions e Transfer extracts to microcentrifuge tubes and centrifuge for 20 min at 13 000 rpm 4 C e Transfer supernatant to a clean tube and store at 70 C Note If the supernatant appears to be cloudy transfer the RayBio L Series Rat Antibody Array L 90 Protocol 6 supernatants to a clean tube centrifuge again at 13 000 rpm for 20 minutes at 2 8 C If the supernatant is still not clear store the lysate at 70 C for 20 minutes Remove from the freezer immediatelv centrifuge at 13 000 rpm for 20 minutes at 2 8 C B Handling the glass slides e The microarrav slides are delicate Please do not touch the arrav surface with pipette tips forceps or vour fingers Hold the slides bv the edges oniv e Handle the slides with powder free gloves and in a clean environment e Do not remove the glass slide from the chamber assembly until step 19 and take great care not to break the glass slide when doing so e Remove reagents sample bv gentiv applving suction with a pipette to corners of each chamber Do not touch the printed area of the array only the sides RavBio L Series Rat Antibodv Arrav L 90 Protocol q C Lavout of Rat L 90 Glass Slide D Two or four identical sub arravs on one slide 2 Subarray 4 Subarray
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11. Completely cover arrays with solution RayBio L Series Rat Antibody Array L 90 Protocol 23 VIII Selected References 1 Christina Scheel et all Paracrine and Autocrine Signals Induce and Maintain Mesenchvmal and Stem Cell States in the Breast Cell 2011 145 926 940 Lin Y Huang R Chen L et al Profiling of cytokine expression by biotin labeled based protein arrays Proteomics 2003 3 1750 1757 Huang R Jiang W Yang J et al A Biotin Label based Antibody Array for High content Profiling of Protein Expression Cancer Genomics Proteomics 2010 7 3 129 141 Liu T Xue R Dong L et al Rapid determination of serological cytokine biomarkers for hepatitis B virus related hepatocellulare carcinoma using antibody arrays Acta Biochim Biophys Sin 2011 43 1 45 51 Cui J Chen Y Chou W C et al An integrated transcriptomic and computational analysis for biomarker identification in gastric cancer Nucl Acids Res 2011 39 4 1197 1207 Jun Zhong et all Temporal Profiling of the Secretome during Adipogenesis in Humans Journal of Proteome Research 2010 9 5228 5238 RavBio L Series Rat Antibody Array L 90 Protocol 26 7 Chowdurv UR Madden BJ Charlesworth MC Fautsch MP Proteomic Analysis of Human Aqueous Humor Invest Ophthalmol Visual Sci 2010 51 10 4921 4931 8 Wei Y Cui C Lainscak M et al Type specific dysregulation of matrix metalloproteinases and their tissue inhibitors
12. RayBio Label Based L Series Rat Antibody Array 90 L 90 Patent Pending Technology User Manual Revised May 14 7 2015 For the simultaneous detection of the relative expression of 90 L 90 rat proteins in serum plasma cell culture supernatants cell tissue lysates or other body fluids L Series Rat Antibody Array L 90 Cat AAR BLG 4 4 Sample Kit Cat AAR BLG 8 8 Sample Kit Please read manual carefully before starting experiment RayBiotech Inc A the protein array pioneer company Your Provider for Excellent Protein Array Systems and Services 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 RavBiotech Inc TABLE OF CONTENTS I Introduction and How It Works II Materials Provided ssssesirzzonresotesjit arcos nas A Storage Recommendati0DS oooooccccconcnncoos B Additional Materials Required III Overview and General Considerations A Preparation and Storage of Samples B Handling the Glass Slides C Glass Slide Lavout nn D Incubation and Washes ooocccccoccccco MW A es E A Dialysis of Sample 0c eeeceeeeeeeeeees B Biotin Labeling of Sample oo o C Drying of the Glass Chip D Blocking and Incubations nee E Fluorescence Detection 0c cece eaeeee
13. ing serum concentrations as low as 0 2 When testing serum containing media we strongly recommend testing an uncultured media blank for comparison with sample results 2 Extracting Protein from Cells e For attached cells remove supernatant from cell culture wash cells twice with cold 1X PBS For suspension cells pellet the cells by centrifuging using a microcentrifuge at 1500 rpm for 10 min e Make sure to remove any remaining PBS before adding 1X RayBio L Series Rat Antibody Array L 90 Protocol 3 Cell Lvsis Buffer 2X Cell Lvsis Buffer should be diluted 2 fold with deionized or distilled water Solubilize the cells at 2x10 cells ml in 1X Cell Lvsis Buffer e Pipette up and down to resuspend cells and rock the Ivsates gently at 2 8 C for 30 minutes Transfer extracts to microfugetubes and centrifuge at 13 000 rpm for 10 min at 2 8 nC e Transfer supernatant to a clean tube Determining cell lylate concentration using a total protein assay BCA Protein Assay Kit Pierce Prod 23227 Aliquot the lysates and store at 70 C Note If the supernatant appears to be cloudy transfer the supernatants to a clean tube centrifuge again at 13 000 rpm for 20 minutes at 2 8 C If the supernatant is still not clear store the lysate at 70 C for 20 minutes Remove from the freezer immediately centrifuge at 13 000 rpm for 20 minutes at 2 8 C 3 Extracting Protein from Crude Tissue e Transfer approximate 100 mg crude tissue
14. is procedure Step 3 We recommended using a BCA total protein assay eg Pierce Catalog 4 23227 B Biotin labeling Sample RavBio L Series Rat Antibody Array L 90 Protocol 10 Note Amines e g Tris glvcine and azides quench the biotinvlation reaction Avoid contaminating samples with these chemicals prior to biotinvlation 4 Immediatelv before use prepare 1X Labeling Reagent Brieflv spin down the Labeling Reagent tube Item B Add 100 ul 1X PBS into the tube pipette up and down or vortex slightiv to dissolve the Ivophilized reagent 5 Add 1X Labeling Reagent to dialvzed samples a For labeling cell culture supernatants transfer 180 ul dialvzed sample into a new tube Add 36 ul of 1X Labeling Reagent Solution per 1 mg total protein in dialvzed cell culture supernatant Mix well For example if sample s total protein concentration is 0 5 mg ml you need to add 3 24 ul 1X Labeling Reagent to 180 ul dialyzed sample b For labeling serum or plasma Add 22 ul of 1X Labeling Reagent Solution into a new tube containing 35 ul dialyzed serum or plasma sample and 155 ul Labeling Buffer Item K Note To normalize serum plasma concentrations during biotinylation measure sample volume before and after dialysis Then adjust the volumes of dialyze serum plasma and Labeling Buffer to compensate to keep same total protein amount and total volume For example if serum plasma sample volume increased from 100 ul to 200 ul
15. ly dry the glass slide using a low velocity Nitrogen gas stream or ambiently in a laminar flow hood or similar clean environment Be sure to protect from light E Fluorescence Detection 24 You may proceed immediately to scanning or you may store the slide at 20 C in the Centrifuge Tube provided or at RT and to scan at a later time RayBio L Series Rat Antibody Array L 90 Protocol Note Unlike most Cy3 fluors the Hilvte Plus Fluor 532 used in this kit is verv stable at RT and resistant to photobleaching on completed glass slides However please protect glass slides from temperatures above RT and store them in the dark Do not expose glass slide to strong light such as sunlight or UV lamp Note If vou need to repeat anv of the incubation after finishing the experiment vou must first re assemble the glass slide into the incubation chamber by following step as shown in the figures below To avoid breaking the printed glass slide you may first want to practice assembling the device with a blank glass slide 1 Apply slide to incubation chamber barcode facing upward as in image A below 2 Gently snap one edge of a snap on side as shown in image B 3 Gently press other of side against lab bench and push in lengthwise direction image C 4 Repeat with the other side image D lim 17 RayBio L Series Rat Antibod V Antibodv Arrav Map RayBio L Series Rat Antibody Array L 90 Protocol 1
16. nal intensities between and among array images can be used to determine relative differences in expression levels of each protein between samples or groups Any 21 5 fold increase or lt 0 65 fold decrease in signal intensity for a single analyte between samples or groups may be considered a measurable and significant difference in expression provided that both sets of signals are well above background Mean background 2 standard deviations accuracy 95 RavBio L Series Rat Antibody Array L 90 Protocol 24 VII Troubleshooting Guide Problem Cause Recommendation Weak signal Inadequate detection Check laser power and PMT parameters Inadequate reagent volumes Check pipettors and or improper dilution ensure correct preparation Short incubation times Ensure sufficient incubation time and change sample incubation step to overnight Too low protein concentration Don t make too low dilution in sample Or concentrate sample Improper storage of kit Store kit at suggested temperature A Use more diluted sample Sample is too concentrated Excess of streptavidin Make sure to use the correct amount of streptavidin Inadequate detection Check laser power and PMT parameters Inadequate wash Increase the volume of wash buffer and incubation time Uneven signal Bubbles formed during incubation Avo id bubble formation during incubation Arrays are not completely covered by reagent
17. ons A Preparation and Storage of Samples 1 Preparation of Cell Culture Supernatants e Seed cells at a density of 1x10 cells in 100 mm tissue culture dishes e Culture in complete culture medium for 24 48 hours e Replenish with serum free or low serum medium such as 0 2 FCS FBS serum and then incubate cells again for 748 hours t Recommended using membrane based array if using high serum medium such as 10 FCS FBS the glass slide arrays tend to have extremely high background for high serum containing media samples e To collect supernatants centrifuge at 1 000 g for 10 min and store as lt 1 ml aliquots at 80 C until needed RayBio L Series Rat Antibody Array L 90 Protocol 4 e Measure the total wet weight of cultured cells in the pellet and or culture dish Vou mav then normalize between arrays by dividing fluorescent signals by total cell mass i e express results as the relative amount of protein expressed mg total cell mass Or you can normalize between array by determining cell lylate concentration using a total protein assay BCA Protein Assay Kit Pierce Prod 23227 Note The density of cells per dish used is dependent on the cell tvpe More or less cells mav be required Optimal culture time may vary and will depend on the cell line treatment conditions and other factors t Bovine serum proteins produce detectable signals on the RayBio L Series Rat Antibody Array 90 in media contain
18. ries Rat Antibodv Arrav L 90 Protocol 2 Il Materials Provided A Storage Recommendations Upon receipt the kit should be stored at 20 C until needed Please use within 6 months from the date of shipment After initial use remaining reagents should be stored at 4 C to avoid repeated freeze thaw cycles may be stored for up to 3 months Labeling Reagent Item B should be fresh preparation before use Unused glass slides should be kept at 20 C and avoid repeated freeze thaw cycles may be stored for up to 6 months RayBio L Series Rat Antibody Array 90 ITEM DESCRIPTION Cat AAR BLG 1 4 Cat AAR BLG 1 8 Dialysis Vials B Labeling Reagent Stop Solution 1 vial 50 ul RayBio L series Rat Antibody Array 1 L li AT l EN L 90 Glass Slides 90 Slide 90 Slides Blocking Butter Tboe m bome m Adhesive Plastic Strips SSCS Labeling Buffer A Each slide contains 4 identical subarrays HiLyte Plus 532 Only needed if testing cell or tissue lysates RayBio L Series Rat Antibody Array L 90 Protocol 3 B Additional Materials Required e Distilled or de ionized water e KCI NaCl KH PO and Na HPO e Small plastic or glass containers e Orbital shaker or oscillating rocker e Beaker stir plate and stir bar e 1mltube e Pipettors pipette tips and other common lab consumables e Laser scanner for fluorescence detection list available online e Aluminum foil III Overview and General Considerati
19. s V Antibody Array Map ennenennnnnnnnnnnnnn VI Interpretation of Results seen nn VII Troubleshooting Guide nee VIII Selected References seen neneeese RavBio L Series Rat Antibodv Arrav L 90 Protocol I Introduction Recent technological advances bv RavBiotech have enabled the largest commercially available antibody array to date With the L Series Antibodv Arrav 90 researchers can now obtain a broad panoramic view of cvtokine expression The expression levels of 90 rat target proteins can be simultaneously detected including cvtokines chemokines adipokine growth factors angiogenic factors proteases soluble receptors soluble adhesion molecules and other proteins in cell culture supernatants serum and plasma The first step in using the RayBio L Series Rat Antibody Array 90 is to biotinvlate the primarv amine of the proteins in serum or plasma samples cell culture supernatant cell Ivsate or tissue Ivsate The glass slide arravs are then blocked just like a Western blot and the biotin labeled sample is added onto the glass slide which is pre printed with capture antibodies and incubated to allow for interaction of target proteins Streptavidin conjugated fluorescent dve Cv3 equivalent is then applied to the arrav Finallv the glass slide is dried and laser fluorescence scanning is used to visualize the signals Here s how it works E a ZZ signaldetection RavBio L Se
20. to microarray analysis If using cell or tissue lysates start at step 2 Dialysis of sample A Dialysis of Sample Note Samples must be dialyzed prior to biotin labeling Steps 5 7 1 To prepare dialysis buffer 1X PBS pH 8 0 dissolve 0 6 g KCI 24 g NaCl 0 6 g KH PO and 3 45 g Na HPO in 2500 ml de RavBio L Series Rat Antibodv Arrav L 90 Protocol 9 ionized or distilled water Adjust pH 8 0 with 1M NaOH and adjust final volume to 3000 ml with de ionized or distilled water 2 Add each sample into a separate Dialysis Tube Item A Load 200 ul cell culture supernatant or 100 ul cell Ivsates or tissue Ivsate 172 mg ml total protein or 20 ul serum or plasma 80 ul 1X PBS pH 8 5 fold dilution Carefully place Dialysis Tubes into Floating Dialvsis Rack Item L 3 Place Floating Dialysis Rack into 2500 ml dialysis buffer in a large beaker Place beaker on a stir plate and dialvze for at least 3 hours at 4C stirring buffer gently Then exchange the 1X PBS buffer and repeat dialysis for at least 3 h at 4 C Transfer dialvzed sample to a clean eppendorf tube Spin dialvzed samples for 5 min at 10 000 rpm to remove anv particulates or precipitants and then transfer the supernatants to a clean tube Note The sample volume mav change during dialvsis Note Dialvsis procedure mav proceed overnight Note Determine the total protein concentration for cell culture supernatants or cell tissue lysate after dialys
21. tored 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 RavBio is a registered trademark of RavBiotech Inc HyLite Plus is a trademark of Anaspec Inc e GenePix is a registered trademark of Molecular Devices Inc RayBio L Series Rat Antibody Array L 90 Protocol This product is for research use oniv 2011 RayBiotech Inc RayBio L Series Rat Antibody Array L 90 Protocol 29
22. xposure to light in Steps 19 25 by covering the Glass Slide Assembly with aluminum foil or incubate in dark room 18 Incubate with Cy3 Conjugated Streptavidin at RT for 2 hours with gentle rocking or shaking Note Incubation may be done overnight at 4 C 19 Decant the solution and disassemble the glass slide from the incubation frame and chamber Disassemble the device by pushing clips outward from the side as shown below Carefully remove the glass slide from the gasket Note Be careful not to touch the printed surface of the glass slide which is on je ON 3 the same side as the barcode RavBio L Series Rat Antibody Array L 90 Protocol 15 20 21 22 23 Note Gentiv place the glass slide into 30 ml Centrifuge Tube Item M Add enough 1X Wash Buffer to cover the entire glass slide Wash with gentle rocking or shaking for 10 min Remove the wash buffer Repeat 2 times for a total of 3 washes Repeat step 20 this time with 1X Wash Buffer ll Repeat one time for a total of two washes for 5 min per wash Finally wash the glass slide with 30 ml of de ionized or distilled water for 5 min Remove glass slide and decant water from Centrifuge Tube Remove excess liquid from Centrifuge Tube and place glass slide into the tube Centrifuge at 1 000 rpm for 3 minutes to remove water droplets Make sure the finished glass slide is completely dry before scanning or storage Alternatively you may gent
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