Manual - RayBiotech, Inc.
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1. 3 zl 5 8 3 N 5l a Z 2 a a e 3 S l a a o al sl zl S Sl s s s s S 5 s al a a aj a ej aj 3 S s 8 3 n n n 1 A n 5 E gt m 3 J e mi om al n ho a 8 8 a lt E a sl 3 3 gt 4 o ules e gl s al 3 o 2 s 3 3 8 2 a sl g mm a 2 al 8 5 e 2 c kal 3 8 8 lt n n sl s a al 3 el g 212 s JE o n sl sl el el l 8 a a 3 sl s 9 s z 3 m ol g Y 8 8 8 a E o 33 I e g 3 S amp 2 hE Li a al 8 gl 8 o 8 8 313 c si 5 5 5 5 of a 5 o kai S 3 sl a a 5 ka s a a a 5 Eli 5 s 5 3 3 3 3 a 5 O RE e 5 5 a a l 4 m S s 8 sla gt EIS G a S s al a a 5 am ma al m sl of nl 14 RayBio L Series Rat Antibody Array 90 Protocol RayBio L Series Rat Antibody Array 90 L 90 List far Je fe et europa C O fea JJ ARA 92 NOR ga TH ANNIE de do IT Y MANN Bank ha j8lak ff f _ 94__Osteopontin SPPT Bo Mo J s Ba le pia s PDGFAA e Mo j be Bam e s e _ Prolactink zoo Ben od lek f e pe fs lace O 8 Jawna he eveek J fe db e ammes e e eS A ae FADD O o SS raton TE Fas Uranio sph E epa 12 JAMPK alphat Ol ____ FasINFRSF6 72__ inuslin O t02___ TGF betat___ 13 Jer rebao fe TEE POS MEF eta gt moo BONE 44____ Follostatin ike 1 FSL1 _ a2. Leptin OB fpo TGFbetas 15 beta Catenin 45 Fracan f s JO fos Tnrombospondin me basic FGF_ j fGFRalpnar gt fe ESaleomcnea foe ME2 _ mz jpetaNGE j jaz Jeff alpnaz2 O mera O ho MP me Jeca e leme pe me ho fme a___ ebi0s___ 49 __ Growth Hormone fo we fho m i 20 CNC 2aiphabeia 50 Growth Hormone A leo WIP talpha___ mo TIR 210 e S i S e e S MP O Fan 2 Jene e heawmcosa O fe MPsapa O m2 amO O 2a CNTE Ralpha ___ ga RT mes Ch ln 2a fesk lsa insun Degrading Enayme ea mmP2 ma Ubin las jexcn j jss JjIEN ganma Io ds MM ds NEGF O Be JEGFR e mama fe MuSkK o pe VERS 27 EO O Z s7____ Btank S 7 E ge lek be e TO T e Poo Polk O eg de dk A j2 Bo Ba O lo le O o f o TEE po EE 15 RayBio L Series Rat Antibody Array 90 Protocol VI Interpretation of Results The following images show the RayBio L Series Rat Antibody Array 90 captured using a chemiluminescence imaging system UVP Biolmaging Systems To obtain optimal results it is suggested to try several different exposure times until the best one is determined Then by comparing the signal intensities relative expression levels of the target proteins can be made The intensities of signals can be quantified by densitometry Anti HRP P 1a P 2a P 3a and anti streptavidin P 1b P 2b P 3b will produce positive control signals which can be used to id
3. deposit into the 50 ml conical collection tube Store at 80 C until needed Discard the Spin Column after use 10 RayBio L Series Rat Antibody Array 90 Protocol D Blocking and Incubation 7 Place each membrane printed side up into a Plastic Incubation Tray provided 1 membrane per tray Note The printed membrane will have a mark in the upper left corner of the membrane 8 Add 2 5 ml of Blocking Buffer Item F to each membrane and cover with the lid Incubate at room temperature with gentle shaking for 1 hour 9 Aspirate Blocking Buffer from each tray Add 2 5 ml of diluted or undiluted sample onto each membrane and cover with the lid Incubate at room temperature with gentle shaking for 2 hours Note 1 It is recommended to use 2 5 ml of 5 fold diluted biotin labeled cell culture supernatant Dilute sample using Blocking Buffer Note 2 The concentration of sample used depends on the abundance of proteins The samples can be concentrated if the overall signals are too weak If the overall signals are too strong the sample can be diluted further Note 3 Incubation may be done at room temperature with gentle shaking for 2 hours or overnight at 4 C 11 RayBio L Series Rat Antibody Array 90 Protocol 10 Dilute 20X Wash Buffer 1 with deionized or distilled water to prepare the 1X Wash Buffer 1 Aspirate the samples from each tray and then wash by adding 3 ml of 1X Wash Buffer at room temp
4. 6 VII Troubleshooting Guide UEUUUE U U LjUEUEjU 18 VIII Reference L St EE E eke 19 RayBio L Series Rat Antibody Array 90 Protocol I Introduction Recent technological advances by RayBiotech have enabled the largest commercially available antibody array to date With the RayBio L Series Rat Antibody Array 90 researchers can now obtain a broad panoramic view of cytokine expression The expression levels of 90 rat proteins can be simultaneously detected including cytokines chemokines adipokine growth factors angiogenic factors proteases soluble receptors soluble adhesion molecules and other proteins in cell culture supernatants The first step in using the Rat L 90 is to biotinylate the primary amine of the proteins in the sample The membrane arrays are then blocked similar to a Western blot and the biotin labeled sample is added onto the membrane array which is pre printed with capture antibodies and incubated to allow for interaction of target proteins After incubation with HRP Conjugated Streptavidin the signals can be visualized by chemiluminescence 2 RayBio L Series Rat Antibody Array 90 Protocol lI Materials Provided A Storage Recommendations Upon receipt Box 1 should be stored at 20 C and Box 2 should be stored at 4 C The kit must be used within 6 months from the date of shipment After initial use Blocking Buffer Stop Solution HRP Conjugated Streptavidi
5. A 2 5 3 0 ml of sample per vial for dialyzing Carefully place all Dialysis Vials into the Floating Rack 3 Place the 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 occasionally gently stirring the dialysis buffer Then exchange the dialysis buffer with fresh buffer and repeat dialysis for at least 3 hours at 4 C Transfer dialyzed samples into a clean eppendorf tube Centrifuge dialyzed samples for 5 minutes at 10 000 rpm to remove any particulates or precipitates and then transfer and combine each sample into one clean eppendorf tube Mix well by gently pipetting Note The sample volume may change during dialysis 8 RayBio L Series Rat Antibody Array 90 Protocol Note Dialysis procedure may proceed overnight C Biotin labeling of Sample Avoid contamination with any solution containing amines i e Tris glycine as well as azides during the biotinylation process 4 Immediately before use prepare 1X Labeling Reagent by briefly centrifuging down the Labeling Reagent vial Item B and add 100 ul 1X PBS pH 8 0 into the vial Pipette up and down or vortex briefly to dissolve the powder 5 Add an appropriate amount of 1X Labeling Reagent into the tube containing the sample and immediately mix the reaction solution Incubate the reaction solution at room temperature for 30 minutes with gentle shaking Gently tap the tube to mix the rea
6. RayBio Label based L Series Rat Antibody Array L 90 Membrane Kit Patent Pending Technology User Manual Revised May 14 2015 For the simultaneous detection of the relative expression of 90 L 90 rat proteins in cell culture supernatants Cat AAR BLM 1 2 2 Sample Kit Cat AAR BLM 1 4 4 Sample Kit Please read manual carefully before starting experiment RayBiotech Inc Hil 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 RayBiotech Inc TABLE OF CONTENTS k WATS HO s dan How It WV OK Sins II Materials Provided A Storage Recommendatione B Additional Materials Required III Overview and General Considerations A Handling Array Membranes B Incubation of Antibody Array IV PROTOCOL KEJ taria toilette N nu Un UN UN U U U N A Preparation of Samp ee B Dialysis of Sample 8 C Biotin labeling Sample 9 D Blocking and Incubation 11 E Del CL O Metatron 12 V Antibody Array Map uieeeeere 14 VI Interpretation of Resultes 1
7. ction solution every 5 minutes Use 7 2 ul of 1X Labeling Reagent for labeling 1 mg of total protein in samples For example if sample s total protein concentration is 0 5 mg ml you need to add 10 8 ul 1X Labeling Reagent to 3 ml dialyzed sample Note The total protein concentration needs to be determined if the sample volume changes after dialysis or if the total protein concentration was determined before the dialysis step 6 Add 5 ul Stop Solution Item D into the reaction solution and 9 RayBio L Series Rat Antibody Array 90 Protocol then use the Spin Column Item J to remove any unbound biotin a Twist off the bottom closure of the Spin Column and loosen the cap but keep the cap on Place the Spin Column into a 50 ml conical collection tube b Centrifuge the Spin Column at 1 000 g for 3 minutes to remove storage solution Note The resin should appear compacted after centrifugation c Add 5 ml 1X PBS pH 8 0 into the Spin Column and centrifuge at 1 000 x g for 3 minutes to remove the 1X PBS Repeat an additional 2 times to wash the Spin Column d Place the Spin Column in a new 50 ml conical collection tube and slowly load 3 5 ml of sample to the center of the compact resin bed Note The maximal sample volume is 4 ml for each Spin Column Do not load over 4 ml of sample into a Spin Column e Centrifuge the Spin Column at 1 000 x g for 3 minutes The sample should filter through the resin and
8. entify the orientation and help normalize the results from different arrays being compared Antibody affinity to its target varies significantly between antibodies The intensity detected on the array with each antibody depends on this affinity therefore signal intensity comparison can be performed only within the same antibody antigen system and not between different antibodies The RayBio Analysis Tool is a program specifically designed for analysis of RayBio L Series Rat Antibody Antibody Arrays This tool will not only assist in compiling and organizing your data but also reduces your calculations to a copy and paste Call RayBiotech Inc at 770 729 2992 for ordering information 16 RayBio L Series Rat Antibody Array 90 Protocol L 90 Membrane Image 17 RayBio L Series Rat Antibody Array 90 Protocol VII Troubleshooting Guide Weak signal or no 1 Taking too much time 1 The whole detection process must be signal for detection completed in 30 min 2 Film developer does 2 Fix film developer not work properly 3 Did not mix HRP 3 Mix tube containing HRP Conjugate streptavidin well before Streptavidin well before use since use precipitates may form during storage 4 Sample is too dilute 4 Increase sample concentration 1 Check if there were any contamination with 5 Other any solution containing amines in biotin labeling step 2 Slightly increase HRP concentrations 3 Work as quic
9. erature with gentle shaking 5 min per wash Repeat the wash 2 more times for a total of 3 washes 11 Aspirate the 1X Wash Buffer 1 from each tray Dilute 20X Wash Buffer 2 with deionized or distilled water to prepare the 1X Wash Buffer 2 Wash 3 times with 3 ml of 1X Wash Buffer 2 at room temperature with gentle shaking 12 Aspirate the 1X Wash Buffer 2 from each tray Dilute the 500X HRP Conjugated Streptavidin with Blocking Buffer to prepare the 1X HRP Conjugated Streptavidin Add 2 5 ml of 1X HRP Conjugated Streptavidin to each membrane Note Ensure that the vial containing the 500X HRP Conjugated Streptavidin is mixed well before use as precipitation can form during storage 13 Incubate at room temperature with gentle shaking for 2 hours Note incubation may be done at 4 C for overnight 14 Wash as directed in steps 10 and 11 E Detection Do not let the membrane dry out during detection The 12 RayBio L Series Rat Antibody Array 90 Protocol detection process must be completed within 40 minutes without stopping 15 For detection of 2 membranes add 2 5 ml of Detection Buffer C and 2 5 ml of Detection buffer D into a tube and mix both solutions Drain off excess wash buffer Place membrane antibody side up symbol is marked in the top left corner of each membrane on a clean plastic plate or its cover provided in the kit Pipette 2 ml of the mixed Detection Buffers on to each membrane and incubate a
10. es 2011 39 4 1197 1207 6 Jun Zhong et all Temporal Profiling of the Secretome during Adipogenesis in Humans Journal of Proteome Research 2010 9 5228 5238 7 Chowdury UR Madden BJ Charlesworth MC Fautsch MP 19 RayBio L Series Rat Antibody Array 90 Protocol 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 in end stage 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 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 20 RayBio L Series Rat Antibody Array 90 Protocol RayBio i
11. kly as possible after mix Detection Buffer C and D 4 Expose film for overnight to detect weak signal Uneven signal 1 Bubbles formed 1 Remove bubbles during incubation during incubation 2 Membranes were not 2 Completely cover membranes with solution completely covered by solution High background 1 Exposure time 1 Decrease exposure time 1s too long 2 Membranes 2 Completely cover membranes with solution dry out during during experiment experiment Cover tray w lid 3 Sample is too 3 Dilute sample concentrated 18 RayBio L Series Rat Antibody Array 90 Protocol 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 2 Lin Y Huang R Chen L et al Profiling of cytokine expression by biotin labeled based protein arrays Proteomics 2003 3 1750 1757 3 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 4 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 5 Cui J Chen Y Chou W C et al An integrated transcriptomic and computational analysis for biomarker identification in gastric cancer Nucl Acids R
12. n Detection Buffers C and D 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 The Array Membrane should be kept at 3 RayBio L Series Rat Antibody Array 90 Protocol 20 C and avoid repeated freeze thaw cycles may be stored for up to 6 months Box 1 store at 20 C TEM DESCRIPTION Cat AAR BLM 1 2 Cat AAR BLM 1 4 Labeling Reagent Stop Solution 1 vial 50 ul 2 membranes 4 membranes E RayBio L Series Rat 90 L 90 L 90 Antibody Array Membranes r Blocking Buffer 1 vial 30 ml 2 vials 30 ml ea 500X HRP Conjugated Veen 1 vial 100 ul 2 vials 100 ul ea Streptavidin Concentrate IK Detection Buffer C 1 vial 10 ml 1 vial 10 ml L Detection Buffer D 1 vial 10 ml 1 vial 10 ml Other Kit Components Plastic Sheets Box 2 store at 4 C TEM 4 vial T vial 30 mi 1 vial 30 mi Spin Columns 4 columns N A Plastic Incubation Trays w lid 1 tray Floating Dialysis Rack 1 rack RayBio L Series Rat Antibody Array 90 Protocol B Additional Materials Required e 1X PBS pH 8 0 e Shaker e 2 5 ml tube e 50 ml conical collection tubes e Distilled water e Kodak X Omat AR film REF 165 1454 and film processor or Chemiluminescence imaging system e large beaker e stir plate e Eppendorf tube Ill Overview and General Considerations A Handling Array Membrane
13. n lt 1 ml aliquots at 80 C until needed 5 Measure the total wet weight of the cultured cells in the pellet and or culture dish 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 Normalization can also be done between arrays by determining the total protein concentration using a total protein assay RayBiotech recommends the Pierce BCA Protein Assay Kit cat 23227 Note The density of cells per dish used is dependent on the cell type More or less cells may be required but should be determined empirically Optimal culture time may be different and depends on cell lines treatment conditions and other factors Bovine serum proteins produce detectable signals on the RayBio L Series membrane arrays at concentrations as low as 0 2 When testing serum containing media it is 7 RayBio L Series Rat Antibody Array 90 Protocol recommended test an uncultured media blank sample for comparison with sample results B Dialysis of Sample Note Samples must be dialyzed prior to biotin labeling Steps 5 7 1 Prepare dialysis buffer 1X PBS by dissolving 0 6 g KCI 24 g NaCl 0 6 g KH PO and 3 45 g Na HPO in 2500 ml de ionized or distilled water Adjust to a pH of 8 0 with 1M NaOH and adjust final volume to 3000 ml with de ionized or distilled water 2 Load each sample into a separate Dialysis Vials Item
14. s e Always use forceps to handle membranes and grip the membranes by the edges only e Never allow membranes to dry during the experiment e Avoid touching membranes with hands or any sharp tools B Incubation e Completely cover membranes with sample or buffer during incubation and cover Plastic Incubation Tray with lid to avoid drying e Avoid foaming during incubation steps e Perform all incubation and wash steps under gentle rotation 5 RayBio L Series Rat Antibody Array 90 Protocol e Several incubation steps such as step 3 in page 10 sample incubation or step 7 in page 11 HRP Conjugated Streptavidin incubation may be done at 4 C for overnight IV Protocol Layout of L 90 Array Membrane 2 5 cm 30 columns x 8 rows Assay Diagram ti 1 Preparation of sample 6 2 Dialysis of sample 3 Determination of protein concentration 4 Biotinylation of sample Li hu 5 Removal of biotin j by spin filter 6 proceed to microarray analysis EN RayBio L Series Rat Antibody Array 90 Protocol A Preparation of Samples 1 Seed cells at a density of 1x10 cells in 100 mm tissue culture dishes 2 Culture in complete culture medium for 24 48 hours 3 Replenish with serum free or low serum medium such as 0 2 FCS FBS and then re incubate cells for 48 hours 4 Collect the cell culture supernatant and centrifuge at 1 000 g for 10 minutes and store i
15. s the trademark of RayBiotech Inc This product is intended for research only and is not to be used for clinical diagnosis These products may not be resold modified for resale or used for manufacture of commercial products without express 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 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 This product is for research use only 2004 RayBiotech Inc 21 RayBio L Series Rat Antibody Array 90 Protocol
16. t room temperature for 2 minutes with gentle shaking Ensure that the detection mixture is evenly covering the membrane without any air bubbles 16 Gently place the membrane with forceps antibody side up on a plastic sheet provided and cover the membrane with another plastic sheet Gently smooth out any air bubbles Avoid using pressure on the membrane Work as quickly as possible 17 The signal can be detected directly from the membrane using a chemiluminescence imaging system or by exposing the array to x ray film we recommend using Kodak X Omat AR film with subsequent development Expose the membranes for 40 seconds Then re expose the film according to the intensity of signals If the signals are too strong background too high reduce exposure time eg 5 30 seconds If the signals are too weak increase exposure time eg 5 20 min or overnight Or re incubate membranes overnight with 1X HRP Conjugated Streptavidin and repeat detection on the second day 13 RayBio L Series Rat Antibody Array 90 Protocol 18 Save membranes at 20 C to 80 C for future reference V Antibody Array Map RayBio L Series Rat Antibody Array 90 Maps if needed larger versions of these maps can obtained by contacting technical support at 770 729 2992 or techsupport raybiotech com ol z l zl zl z a 2 a 3 3 5 3 7 a 5 a 5 al
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