RayBio Biotin Label-based Human Obesity Antibody Array 1
Contents
1. VINE I TT T RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol oo N N N I Introduction The area of obesity research is getting hotter ever over the past years One of the key driving force is that adipose tissue is found no longer to be an inert energy storage organ but is emerging as an active participant in regulating physiological and pathologic processes Many soluble factors have been identified from the adipose tissue and are so called as adipocytokines or adipokines Some of the adipokines are mainly produced by the adipose tissue like leptin and resistin while others are also synthesized in other tissues like TNF alpha IL 6 MCP 1 and IL 1 Because all of these factors can act in an autocrine paracrine or endocrine manner in the organisms adipokines are thought to serve as mediators linking obesity inflammation immunity and other obesity related diseases Recent technological advances by Raybiotech have enabled the largest commercially available antibody array to date With the L Series Human Obesity Antibody Array 1 researchers can now obtain a broad panoramic view of adipokine expression The expression levels of 182 human target proteins can be simultaneously detected in cell culture supernates and serum Furthermore an internal control 15 used to monitor the whole process including biotin labeling so this massive array will accurately refle2. 27 udsen HSL HSL Fos 9 551 WHS Was TuS NL udis NL NL EL vupuodsoquog zupuodsoqwos z uipuodsoqwosy 9 91 9 91 vuol vuol xoa 77 301 90015 unsisou ras S31NVu AM u VY 490d 88 490d 88 3904 Ouvd 0 50 50 unaeuoaso un2euoeiso 0 WSO nso guxeo 8 uxo or 2 7 HSN Srann erann trann mm 277 acum 6 an an suouuoH Burzum HT Burzujemi H1 U yundan 50d 2 2504 180d 180d 8 ocu oru uso ru 1 222 2 L OSH IOSH 20 2049 2089 euoiuieinio esepixcied euojuieinio 9 uuo mo weno unano ououuoH ymo H auounioH uwo19 1 un2ejen k HS4 5304 9494 01 494 01 454 5 SLNN3 o3 X493 393 EI a duo epnded o epnded o ALND Sor ENW shana s ana s ana rana rana sana sana rana g Tdv VILdONV raon Fd NY Jope4 ex Buy CENNI 1ru
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4. Samples must be dialyzed prior to biotin labeling Steps 5 7 1 prepare dialysis buffer 1X PBS pH 8 0 dissolve 0 6 g KCI 24 g NaCl 0 6 g and 3 45 g Na HPO in 2500 ml de 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 Dialyzer Tube D Tube Item A Load 200 ul cell culture supernate or 20 ul serum or plasma 80 ul 1X PBS pH 8 5 fold dilution Carefully place Dialyzer Tubes into D Tube Floating Rack Item L RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 8 3 Place D Tube Floating Rack into gt 500 ml dialysis buffer in a large beaker Place beaker on a stir plate and dialyze for at least 3 hours at 4 C stirring buffer gently Then exchange the 1X PBS buffer and repeat dialysis for at least 3 h at 4 C Transfer dialyzed sample to a clean eppendorf tube Spin dialyzed samples for 5 min at 10 000 rpm to remove any particulates or precipitants and then transfer the supernates to a clean tube Note The sample volume may change during dialysis Note Dialysis procedure may proceed overnight Biotin labeling Sample Note Amines e g Tris glycine and azides quench the biotinylation reaction Avoid contaminating samples with these chemicals prior to biotinylation 4 Immediately before use prepare 1X Labeling Reagent Briefly spin down the Labeling Reagent tube Item B
5. 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 without endometriosi 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 Biotin Label based 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 RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 23 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 stored properly In the event of any defec
6. Add 100 ul 1X PBS into the tube pipette up and down or vortex slightly to dissolve the lyophilized reagent 5 Add IX Labeling Reagent to dialyzed samples a For labeling cell culture supernates transfer 180 ul dialyzed sample into a new tube Add 36 ul of IX Labeling Reagent Solution per 1 mg total protein in dialyzed cell culture supernate Mix well RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 9 Note Determine the total protein concentration immediately prior to biotin labeling Step 5 We recommended using a BCA total protein assay eg Pierce Catalog 23227 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 Serum Buffer Item K Note To normalize serum plasma concentrations during biotinylation measure sample volume before and after dialysis Then adjust the volumes of dialyzed serum and Serum Buffer to compensate For example if serum plasma sample volume increased from 100 ul to 200 ul add 70 ul dialyzed serum and 120 ul Serum Buffer 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
7. Antibody Array e Cover incubation chamber with adhesive film Item J to prevent evaporation during incubation or wash steps particularly those lasting 2 hours or longer e During incubation and wash steps avoid foaming and be sure to remove all bubbles from the sub array surface e Perform all incubation and wash steps under gentle rotation or rocking motion 0 5 to 1 cycle s e Wash steps in Wash Buffer II and all incubation steps may be performed overnight at 4 C RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 5 e Avoid cross contamination of samples to neighboring wells To remove Wash Buffers and other reagents from chamber wells you may invert the Glass Chip Assembly to decant and aspirate the remaining liquid e Unlike most Cy3 fluors the HiLyte Plus Fluor 532 used in this kit is very stable at RT and resistant to photobleaching on completed glass chips However please protect glass chips from strong light and temperatures above RT C Layout of Array Glass Chip 2 Subarray 4 Subarray Blank RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 6 D Preparation of Cell Culture Supernates 1 Plate cells at a density of 1x10 cells in 100 mm tissue culture dishes 2 Cultured in complete culture medium for 24 48 hours 3 Replenish with serum free or low serum medium such as 0 2 FCS FBS serum and then incubate cells again for 48 hours F 4 To collect
8. proceed with the assay Dry the Glass Chip 8 Remove the package containing the Glass Chip Assembly Item E from the freezer Place unopened package on the benchtop for approx 15 min and allow the Glass Chip Assembly to equilibrate to room temperature RT 9 Open package and take the Glass Chip Assembly out of the RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 10 sleeve Do not remove the Glass Chip from the chamber assembly Place glass chip assembly in laminar flow hood or similar clean environment for 1 2 hours at RT Note Protect the chip from dust or others contaminants Blocking and Incubation of Antibody Array Note Glass chip should be completely dry before adding Blocking Buffer to wells 10 Block sub arrays by adding 400 ul of Blocking Buffer Item F into each well of Glass Chip Assembly and incubating at RT for 30 min Remove any bubbles on the array surfaces 11 Immediately prior to sample incubation spin biotin labeled samples for 5 min at 10 000 rpm to remove any 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 supernates or 20 fold for serum plasma 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 s
9. supernates centrifuge at 1 000 g for 10 min and store as lt 1 ml aliquots at 80 C until needed 5 Measure the total wet weight of cultured cells in the pellet and or culture dish You may then normalize between arrays by dividing fluorescent signals by total cell mass 1 express results as the relative amount of protein expressed mg total cell mass you nomalize 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 type More or less cells may be required Optimal culture time may be different and depends on your cell lines treatment conditions and other factors Bovine serum proteins produce detectable signals on the RayBio amp Human Obesity Label based Antibody Array in media containing 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 RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 7 V Protocol Assay Diagram 1 Cell Culture Supernates 2 Serum or plasma 1 Preparation of sample 2 Dialysis of sample 2 Dialysis of sample 3 Determination of protein concentration 3 Biotinylation of sample 4 Biotinylation of sample 5 Dialysis of biotinylated sample Dialysis of Sample Note
10. DI 1 2 or 4 sub arrays for AAH BLG ADI 1 4 Blocking Buffer Item F 8 ml 20X Wash Buffer I Item G 30 ml 20X Wash Buffer II Item H 30 ml HiLyte Plus 532 Streptavidin conjugated Fluorescent dye Item I Cy3 equivalent 1 tube per glass chip Adhesive film Item J Serum Buffer Item K 8 ml D Tube Floating Rack Item L 30 ml Centrifuge tube Item M Additional Materials Required Distilled or de ionized water KCl NaCl KH PO and Na HPO Small plastic or glass containers Orbital shaker or oscillating rocker Beaker stir plate and stir bar 1 ml tube Pipettors pipette tips and other common lab consumables Laser scanner for fluorescence detection list of compatible scanners available at http www raybiotech com resources asp Aluminum foil RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 4 IV Overview and General Considerations A Handling glass chips e The microarray slides are delicate Please do not touch the array surface with pipette tips forceps or your fingers Hold the slides by the edges only e Handle the slides with powder free gloves and in a clean environment e Do not remove the glass chip from the chamber assembly until step 19 and take great care not to break the glass chip when doing so e Remove the final buffer by gently applying suction with a pipette to corners of each chamber Do not touch the printed area of the array only the sides B Incubation of
11. RayBio Biotin Label based Human Obesity Antibody Array 1 For the Simultaneous Detection of the Relative Expression of 108 Human Proteins in Cell Culture Supernates Serum or Plasma AAH BLG ADI 1 2 and AAH BLG ADI 1 4 User Manual Revised Aug 25 2011 Please read manual carefully before starting experiment RayBiotech Inc ds the protein array pioneer company As the Protein Array Pioneer Company Excellence and Innovation Is Our Goal 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 550 mimm RayBiotech Inc TABLE OF CONTENTS LL 2 espere HOW IU Materials Provided eire etr teet Additional Materials Required IV Overview and General Considerations A Handling Glass B Incubation of Antibody Array C Layout of Glass Chip D Preparation of Samples ca Dialysis Biotin Labeling of Sample Dry the Glass Cp Blocking and Incubation of Antibody Array Fluorescence 2 VI Interpretation of VII Troubleshooting
12. ct the available adipokines in your sample The first step in using the RayBio Biotin label based human obesity antibody array 1 is to biotinylate the primary amine of the proteins in cell culture supernates and serum The biotin labeled sample is then added onto glass chip and incubated at room temperature Fluorescent dye Conjugated Streptavidin cy3 equivalent is used to visualize the signals RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 2 Here s how it works ot Ve 0 7 i Pg of Ww i samples 1 biotin label reagent array support y 4 ob ob ob ob vy biotin labeled samples incubation with labeled streptavidin signal detection II Materials Provided 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 Unused glass chips should be kept at 20 C e Dialysis tube Item A 4 tubes for AAH BLG ADI 1 2 or 8 tubes for AAH BLG ADI 1 4 e Labeling Reagent Item B 1 tube for AAH BLG ADI 1 2 or 2 tubes for AAH BLG ADI 1 4 RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 3 Stop Solution Item D 50 ul RayBio Biotin Label based Human Obesity Antibody Array Glass Chip in Chamber Assembly Item E 2 sub arrays or 4 sub arrays in one glass chip 2 sub arrays for AAH BLG A
13. f you need to repeat any of the incubation after finishing the experiment you must first re assemble the glass chip into the incubation chamber by following step as shown in the figures below To avoid breaking the printed glass chip 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 below 2 Gently snap one edge of a snap on side as shown in image 3 Gently press other of side against lab bench and push in lengthwise direction image C 4 Repeat with the other side image D A RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 15 VI Interpretation of Results A Explanation of Controls Spots 1 2 Positive Control spots 51 POS2 POS3 standardized amounts of biotinylated IgGs printed directly onto the array 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 Steptavidin conjugated Fluor Negative c
14. haking for 2 hours at RT or overnight at 4 C 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 RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 11 diluted further in subsequent experiments If the signal is too weak more concentrated samples can be used Note Avoid the flow of sample into neighboring wells 13 14 15 16 Dilute 20X Wash Buffer I 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 I at RT with gentle rocking or shaking for 5 min per wash Obtain a clean container e g pipette tip box or slide staining jar place the Glass Chip Assembly into the box with sufficient 1X Wash Buffer I to completely cover the entire assembly and remove all bubbles in wells Wash 2 times at RT with gentle rocking or shaking for 10 min per wash Dilute 20X Wash Buffer II Concentrate Item H 20 fold with de ionized or distilled water Decant the Wash Buffer I from each well place the Glass Chip Assembly into the box with sufficient 1X Wash Buffer II to completely cover the entire assembly and remove all bubbles in wells Wash 2 times at RT with gentle rocking or shaking for 5 min per wash Prepare Streptavidin conjugated Fluorescent Dye a Briefly spin down tube containing the Strep
15. ize signal intensities to the Positive Controls To order the Analysis Tool please contact us at 1 770 729 2992 or info Q raybiotech com for more information E Threshold of significant difference in expression RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 18 After subtracting background signals and normalization to Positive Controls comparison of signal intensities between and among array images can be used to determine relative differences in expression levels of each protein between samples or groups Any gt 1 5 019 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 RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 19 sod 1504 2594 2504 80d sod uvaax uvaax 1438d unejsA taag AUNES 493A i can re ET eva 12 ensi 0140 10198 enssit vani vupuodsoquom zi 5 t uecopu s uuotoues vivas vevwas raas raas ws ws 01 0015 oiv 0015 0015 6v ev 015 40015 i bela bed EOIN
16. oftware 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 e g a column labeled as MED532 RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 17 8532 you may need to subtract the background manually 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 PI P y 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 The RayBio Analysis 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 normal
17. ontrol signal intensities are usually very close to background signals in each sub array B Typical results obtained with Biotin Label based Human Obesity Antibody Array 1 The following figure shows the RayBio Biotin label based Human Obesity Antibody Array 1 probed with serum sample The images were captured using a Axon GenePix laser scanner The strong signals in row 8 and the the upper left and lower right corners of each array are Positive Controls which can be used to identify the orientation and help normalize the results between arrays RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 16 If scanned using optimal settings 3 distinct signal intensities will be seen POSI 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 concentrations of the various analytes in your samples try using our Quantibody 9 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 s
18. otocol 21 VIII Reference List 1 Christina Scheel et all Paracrine and Autocrine Signals Induce and Maintain Mesenchymal 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 Chowdury UR Madden BJ Charlesworth MC Fautsch MP Proteomic Analysis of Human Aqueous Humor Invest Ophthalmol Visual Sci 2010 51 10 4921 4931 Wei Y Cui C Lainscak M et al Type specific dysregulation of RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 22 matrix metalloproteinases and their tissue inhibitors in end stage heart failure patients relationshp between MMP 10 and LV
19. rsuejojBuy 1rursusyojfuy 2 dav zaw zaw 8010 30 504 504 2804 2504 1 50 1 50 L s vi EL mn 1 6 g n 9 s v g z i dv 4 poqnguy suryodipy poseq oqe T urorg orgAey 20 RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 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 in sample Don t make too low dilution Or concentrate sample Improper storage of kit Store kit at suggested temperature Sample is too concentrated Use more diluted sample 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 Avo id bubble formation incubation during incubation Arrays are not completely covered by reagent Completely cover arrays with solution RayBio Biotin Label based Human Obesity Antibody Array 1 Pr
20. ss chip Wash with gentle rocking or shaking for 10 min Remove the wash buffer Repeat 2 times for a total of 3 washes RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 13 21 Repeat step 21 this time with 1X Wash Buffer II Repeat one time for a total of two washes for 5 min per wash 22 Finally wash the glass chip with 30 ml of de ionized or distilled water for 5 min Remove glass chip and decant water from Centrifuge Tube 23 Remove excess liquid from Centrifuge Tube and place glass chip into the tube Centrifuge at 1 000 rpm for 3 minutes to remove water droplets Make sure the finished elass chip is completely dry before scanning or storage Note Alternatively you may gently dry the glass chip using a low velocity Nitrogen gas stream or ambiently in a laminar flow hood or similar clean environment Be sure to protect from light Fluorescence Detection 24 You may proceed immediately to scanning or you may store the slide at 20 C in the Centrifuge Tube provided at RT and to scan at a later time Note Unlike most Cy3 fluors the HiLyte Plus Fluor 532 used in this kit is very stable at RT and resistant to photobleaching on completed glass chips However please protect glass chips from temperatures above RT and store them in the dark Do not expose glass chip to strong light such as sunlight or UV lamp RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 14 Note I
21. t 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 RayBio is a registered trademark of RayBiotech Inc HyLite Plus is a trademark of Anaspec Inc GenePix is a registered trademark of Molecular Devices Inc RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 24 This product is for research use only 92011 RayBiotech Inc RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 25
22. tavidin conjugated Fluorescent Dye Item I immediately before use b Add 1000 ul of Blocking Buffer into the tube to prepare a concentrated Streptavidin Fluor stock solution Pipette up and down to mix gently do not store the stock solution for later use RayBio Biotin Label based Human Obesity Antibody Array 1 Protocol 12 c Add 200 ul of Streptavidin Fluor concentrate into tube with 800 ul of Blocking Buffer Mix gently to prepare 1X working dilution 17 Carefully remove Glass Chip Assembly from containter Remove all of Wash Buffer II from the wells Add 400 ul of 1X Streptavidin conjugated Fluorescent dye to each sub array Cover the incubation chamber with adhesive film Note Avoid exposure to light in Steps 19 25 by covering the Glass Chip Assembly with aluminum foil or incubate in dark room 18 Incubate with Streptavidin Fluor 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 chip from the incubation frame and chamber Disassemble the device by pushing clips outward from the side as shown below Carefully remove the glass chip from the gasket Note Be careful not to touch the printed surface of the glass chip gt 1 which is the same side as the ad barcode 20 Gently place the glass chip into 30 ml Centrifuge Tube Item M Add enough 1X Wash Buffer I to cover the entire gla
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