RayBio Biotin Label-based Rat Antibody Array 1
Contents
1. Connexin suppresses human glioblastoma cell growth by down regulation of monocyte chemotactic protein 1 as discovered using protein array technology Ruochun Huang Ying Lin Cheng C Wang Jacob Gano Biaoyang Lin Qian Shi Alton Boynton Jocelyn Burke and Ruo Pan Huang Cancer Res 2002 62 2806 2812 LPS induces the interaction of a transcription factor LPS induced TNF a factor and STAT6 B with effects on multiple cytokines 16 RayBio Biotin Label based Rat Antibody Array 1 Protocol Xiaoren Tang Deborah Levy Marciano Susan E Leeman and Salomon Amar PNAS April 5 2005 vol 102 no 14 5132 5137 7 HIV 1 mediated apoptosis of neuronal cells Proximal molecular mechanisms of HIV 1 induced encephalopathy Yan Xu Joseph Kulkoshy Roger j Pomerantz PNAS 2004 May 4 2004 Vol 101 No 18 8 A novel method for high throughput protein profiling from conditioned media and patient s sera Ruo Pan Huang Ruochun Huang Yan Fan and Ying Lin Ana Biochem 2001 294 1 55 62 RayBio is the trademark of RayBiotech Inc This product is intended for research only and is not to be used for clinical diagnosis Our 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 date2. produce positive control signals which can be used to identify 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 Biotin Label based Antibody Arrays This tool will not only assist in compiling and organizing your data but also reduces your calculations to a 12 RayBio Biotin Label based Rat Antibody Array 1 Protocol copy and paste Call RayBiotech Inc at 770 729 2992 for ordering information RayBio Biotin Label based Rat Antibody List OONOORWND P 1a P 1b P 1c Blank Neg Neg Blank Activin A ACTH ADFP Adiponectin Acrp30 AMPK alpha 1 B7 1 CD80 BDNF beta Catenin basic FGF beta NGF CCR4 CD106 CINC 2 alpha beta CINC 3 CNTF CNTF R alpha CSK CXCR4 EGFR Blank IC 3 IC 2 IC 1 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Blank Blank Blank Blank Blank Blank Blank EG VEGF PK1 E Selectin FADD Fas Ligand TNFSF6 Fas TNFRSF6 FGF BP Follostatin like 1 FSL1 Fractalkine GFR alpha 1 GFR alpha 2
3. 69 89 L9 99 59 r9 9 c9 T9 S SN 95 SS 75 5 55 Is 05 6r 8r Lb 9r st rt p Th Ww or 6 Sc MUR opuepg uel uepg Jwg t 95 ss bs s 55 Is 05 6r sr LY 9r st th tr c 114 or 6 Sc ueg ueg Ue uel Jwg FOI COI CDM weg 9 sc vc c cc Ic 0 6r 8I LI SI I rai I or 6 8 39N SN d Bd T FOI T L fOr yeg 9c sc vc c cc 0c 6r 8I LI or SI I ct 6 8 yug SN Ue I 05 6c 87 Lc 9c vc tc cc Tc 0 SI LI 9T SI I TI n 6 8 L 9 5 t T I eN T Apoqnuy wy poseg oqe T unorgGorg Aes 14 RayBio Biotin Label based Rat Antibody Array 1 Protocol VII Troubleshooting Guide Problem Cause Recommendation Weak signal or no 1 Taking too much time signal for Detection 2 Film developer does not work properly 3 Did not mix HRP streptavidin well before use 4 Sample is too dilute 5 Other Uneven signal 1 Bubbles formed during incubation 2 Membranes were not completely covered by solution High background 1 Exposure to x ray file is too long 2 Membranes were 1 The whole Detection process must be completed in 30 min 2 Fix film developer 3 Mix tube containing HRP Conjugate Streptavidin well before use since precipitates may form during storage 4 Increase sample concentration 1 Reduce blocking concentration by diluting in
4. C 3 RayBio Biotin Label based Rat Antibody Array 1 Protocol Box 1 store at 20 C Labeling Reagent Item B 1 tube for 2 array membranes and 2 for 4 array membranes Internal control Item C 1 tube for 2 array membranes and 2 for 4 array membranes Stop Solution Item D 50 ul RayBio Biotin label based Rat Antibody Array 1 Item E 2 membrane for Cat AAR BLM 1 2 and 4 for AAR BLM 1 4 Blocking Buffer Item F 30 ml for each bottle 1 bottles for 2 array membranes and 2 for 4 array membranes 500X HRP Conjugated Streptavidin Concentrate Item I 100 ul Detection Buffer C Item K 5 ml for 2 membranes and 10 ml for 4 membranes Detection Buffer D Item L 5 ml for 2 membranes and 10 ml for 4 membranes Plastic sheet Box 2 store at 4 C Dialysis tube and Floating Rack Item A 2 tubes for 2 array membranes and 4 for 4 array membranes dialysis tube is from EMD product 20X Wash Buffer I Item G 30m1 20X Wash Buffer II Item H 30m1 Spin Column Item J 2 columns for 2 array membranes and 4 for 4 array membranes Plate 1 plate 4 RayBio Biotin Label based Rat Antibody Array 1 Protocol III Additional Materials Required 1X PBS pH 8 0 Shaker 2 5 ml tube 50 ml conical collection tube Distilled water Kodak X Omat AR film REF 165 1454 and film processor or Chemiluminescence imaging system IV Overview and General Considerations A Handling Array Membranes e A
5. 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 17 RayBio Biotin Label based Rat Antibody Array 1 Protocol Note 18 RayBio Biotin Label based Rat Antibody Array 1 Protocol Note 19 RayBio Biotin Label based Rat Antibody Array 1 Protocol Note 20 RayBio Biotin Label based Rat Antibody Array 1 Protocol This product is for research use only 2008 RayBiotech Inc 21 RayBio Biotin Label based Rat Antibody Array 1 Protocol
6. 1X Wash Buffer II 2 Slightly increase HRP concentrations 3 Slightly increase biotinylate antibody concentrations 4 Expose film for overnight to detect weak signal 1 Remove bubbles during incubation 2 Completely cover membranes with solution 1 Decrease exposure time 2 Completely cover membranes with solution allowed to dry out during during experiment experiment 3 Sample is too concentrated 3 Use more diluted sample 15 RayBio Biotin Label based Rat Antibody Array 1 Protocol VIII Reference List 1 Profiling of cytokine expression by biotin labeled based protein arrays Ying Lin Ruochun Huang Lipai Chen et al Proteomics 2003 3 1750 1757 Proteomic profiling of the cancer microenvironment by antibody arrays Vladimir Knezevic Chidchanok Leethanakul Verena E Bichsel et al Proteomics 2001 1 1271 1278 Antibody microarray profiling of rat prostate cancer sera Antibody screening and identification of potential biomarkers Jeremy C Miller Heping Zhou Joshua Kwekel Robert Cavallo et al The Brian B Haab Proteomics 2003 3 56 63 Smad4 signalling in T cells is required for suppression of gastrointestinal cancer Byung Gyu Kim Cuiling Li Wenhui Qiao Mizuko Mamura Barbara Kasperczak Miriam Anver Lawrence Wolfraim Suntaek Hong Elizabeth Mushinski Michael Potter Seong Jin Kim Xin Yuan Fu Chuxia Deng and John J Letterio Nature 2006 Vol 441 1015
7. GM CSF Growth Hormone Growth Hormone R Hepassocin ICAM 1 CD54 ICK Insulin Degrading Enzyme IFN gamma IL 1 alpha Blank Neg Neg Neg 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 13 IL 1 beta IL 1 R6 IL 1 R rp2 IL 2 IL 3 IL 4 IL 5 IL 6 IL 10 IL 12 IL 23 p40 IL 13 Integrin alpha M beta 2 Insulin IP 10 Leptin OB LIX L Selectin CD62L MCP 1 MDC MIF MIP 1 alpha MIP 2 MIP 3 alpha MMP 13 MMP 2 MMP 8 MuSK Blank Blank Blank Blank 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 ee ee Neuropilin 2 NGFR Orexin A Osteopontin SPP1 PDGF AA Prolactin R RAGE RALT MIG 6 RELM beta Resistin TALIA TGF beta1 TGF beta2 TGF beta3 Thrombospondin TIE 2 TIMP 1 TIMP 2 TIMP 3 TLR4 TNF alpha TRAIL TROY Ubiquitin VEGF VEGF C Blank P 2c P 2b P 2a RayBio Biotin Label based Rat Antibody Array 1 Protocol 924 d ywa MI Su FI err Hr 601 801 LO 901 Sor ror 01 cor 001 66 86 L6 96 56 76 56 56 16 8 Cd 94 Vd WRT YT SI FIT err cu Hr 601 801 LOT 90T sor ror or cor TOT 001 66 86 L6 96 56 76 6 c6 T6 L ueg 98 58 78 8 c8 I8 08 6L 8L LL 9L SL TL L TL IL 0L 69 89 L9 99 59 r9 9 c9 T9 9 quel uel yueg 98 58 78 8 c8 I8 08 6L 82 LL 9L SL bL L TL IL 0L
8. RayBio Biotin Label based Rat Antibody Array 1 For the Simultaneous Detection of the Expression Levels of 90 Rat Proteins in Cell Culture Supernates User Manual Revised Apr 1 2009 Cat AAR BLM 1 2 AAR BLM 1 4 RayBiotech Inc As the Protein Array Pioneer Company Excellence and Innovation Is Our Goal Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 770 206 2393 Web www raybiotech com Email info raybiotech com RayBiotech Inc TABLE OF CONTENTS I JEntrOdUCUOEbe etr editus How Tt Materials Provided eer Pede III Additional Materials Required IV Overview and General Considerations A Handling Array Membranes B Incubation of Antibody Array DE LI cu A Preparation of Samples B Dialysis of Sample C Biotin labeling Sample NNO nr t W WN VI D Blocking and Incubation of Antibody Array E Detection Interpretation of VII Troubleshooting Guide VIII Reference List RayBio Biotin Label based Rat Antibody Array 1 Protocol Nel I Introduc
9. fter centrifugation c Add 5 ml 1X PBS into column centrifuge at 1 000 g for 3 minutes to 1X PBS Repeat additional 2 times to wash the column d Place the column in a new collection tube slowly load the sample to the center of the compact resin bed e Centrifuge the column at 1 000 g for 3 minutes to collect sample Stored at 80 C until testing Discard column after use D Blocking and Incubation 66 1 Place each membrane into the provided tray mark is on the antibody printed side Note The printed side should be facing upward 2 Add 2 5 ml Blocking Buffer and incubate at room temperature for 1 hour to block membranes 3 Decant Blocking Buffer from each container Add 2 5 ml of sample into each array membrane and cover with the lid Incubate at room temperature with gentle shaking for 2 hours Dilute sample using Blocking Buffer Note 1 We recommended using 2 5 ml of 5 fold diluted cell culture supernates which have been biotin labeled 9 RayBio Biotin Label based Rat Antibody Array 1 Protocol Dilute sample using Blocking Buffer Note 2 The amount of sample used depends on the abundance of protein More of the sample can be used if signals are too weak If signals are too strong the sample can be diluted further Note 3 Incubation may be done at room temperature for 2 hours Over night at 4 C Decant the samples from each container and wash 3 times with 3 ml of 1X Was
10. h Buffer I at room temperature with shaking 5 min per wash Dilute 20X Wash Buffer I with deionized or distilled water Decant the 1X Wash Buffer I from each container Wash 3 times with 3 ml of 1X Wash Buffer II at room temperature with gentle shaking Add 2 5 ml of 500 fold diluted HRP conjugated streptavidin e g add 10 ul of HRP conjugated streptavidin to 5 ml of Blocking Buffer to each membrane Note Mix tube containing 500X HRP Conjugated Streptavidin well before use since precipitation may form during storage 7 Incubate at room temperature with gentle shaking for 2 hours Note incubation may be done at 4 C for overnight 10 RayBio Biotin Label based Rat Antibody Array 1 Protocol 8 Wash as directed in steps 4 and 5 E Detection Do not let the membrane dry out during detection The detection process must be completed within 40 minutes without stopping 1 Add 2 5 ml of Detection Buffer C and 2 5 ml of Detection Buffer D into a tube for detecting 2 membranes Mix both solutions Drain off excess wash buffer Place membrane protein side up mark is on the protein side top left corner on a clean plastic plate or its cover provided in the kit Pipette 2 2 ml of the mixed Detection Buffer on to each membrane and incubate at room temperature with shaking for 2 minutes Ensure that the detection mixture is evenly covering the membrane without any air bubbles 2 Gently place the membrane wi
11. lways use forceps to handle membranes and grip the membranes by the edges only e Never allow array membranes to dry during experiments e Avoid touch Array membrane by hand tips or any sharp tools B Incubation e Completely cover membranes with sample or buffer during incubation and cover eight well tray with lid to avoid drying e Avoid foaming during incubation steps e Perform all incubation and wash steps under gentle rotation e Several incubation steps such as step 3 in page 10 sample incubation or step 7 in page 11 HRP streptavidin incubation may be done at 4 C for overnight 5 RayBio Biotin Label based Rat Antibody Array 1 Protocol V Protocol Assay Diagram P s l Preparation of sample 2 Dialysis of sample 3 Determination of protein concentration 4 Biotinylation of sample 5 Removal of biotin by spin filter 6 proceed to microarray analysis A Preparation of Samples The cell culture supernates can be prepared in the following conventional manner To prepare cell culture supernates cell conditioned media cells are plated in 100 mm tissue culture dishes at a density of 1x10 cells per dish The cells are then cultured with complete culture medium for 24 48 hours The complete culture medium is replaced with lower serum medium such as 0 2 FCS serum and then the cells are cultured for 48 hour again once more The supernates are collected centrifuged at 1 000 g aliq
12. ontrol tube Item C before use 7 RayBio Biotin Label based Rat Antibody Array 1 Protocol Add 100 ul 1X PBS pH 8 0 into the Internal Control tube pipette up and down to dissolve the powder Transfer 2 ml dialyzed sample into a new tube Add 40 ul prepared Internal Control into the tube Mix well 2 Immediately before use briefly spin down the Labeling Reagent tube Item B Add 100 ul 1X PBS into the tube pipette up and down or vortex to dissolve the powder to prepare 1 X Labeling Reagent solution 3 Add an appropriate amount of prepared Labeling Reagent into above tube with sample in step 2 mix well immediately Incubate the reaction solution at room temperature for 30 min with gentle shaking Gently tap the tube to mix the reaction solution every 5 min 7 2 ul of IX Labeling Reagent for labeling 1 mg total protein in supernates Note You need to re calculate the total protein concentration if cell culture supernatet volume is changed after dialysis and you measure the total protein concentration before dialysis step 4 Add 5 ul Stop Solution into the above reaction solution and then use the spin column to remove free biotin a Twist off the spin column s bottom closure and loosen the cap Place the column into a 50 ml collection tube b Centrifuge column at 1 000 g for 3 minutes to remove storage solution 8 RayBio Biotin Label based Rat Antibody Array 1 Protocol Note The resin will appear compacted a
13. th forceps protein side up on a piece of plastic sheet mark 1 on the protein side top left corner Cover the array with another piece of plastic sheet Gently smooth out any air bubbles Avoid using pressure on the membrane Work as quickly as possible 3 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 M 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 11 RayBio Biotin Label based Rat Antibody Array 1 Protocol 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 4 Save membranes at 20 C to 80 C for future reference VI Interpretation of Results The following figure shows the RayBio Biotin label based Array I probed with cell culture supernates One important parameter is the background signal To obtain the best results we suggest that several exposures be attempted By comparing the signal intensities relative expression levels of target proteins can be made The intensities of signals can be quantified by densitometry A biotinylated protein and internal control will
14. tion Recent technological advances by Raybiotech have enabled the largest commercially available antibody array to date With the L Series 90 researchers can now obtain a broad view of cytokine expression The expression levels of 90 rat target proteins can be simultaneously detected including cytokines chemokines adipokine growth factors angiogenic factors proteases soluble receptors sobuble adhesion molecules and other proteins in cell culture supernates Furthermore an internal control is used to monitor the whole process including biotin labeling so this massive array will accurately reflect the available cytokines in your sample The first step in using the RayBio Biotin label based rat antibody array is to biotinylate the primary amine of the proteins in cell culture supernates The biotin labeled sample is then added onto array membrane and incubated at room temperature After incubation with HRP streptavidin the signals can be visualized by chemiluminescence 2 RayBio Biotin Label based Rat Antibody Array 1 Protocol II Materials Provided Upon receipt the Box 1 should be stored at 20 C and Box 2 should be stored at 4 C Please use within 6 months from the date of shipment After initial use the Blocking Buffer Stop solution HRP Conjugated Streptavidin Detection Buffer C and D should be stored at 4 C to avoid repeated freeze thaw cycles The Array Membrane and Internal Control should be kept at 20
15. uoted and stored at 80 C until use Meanwhile the cells are also collected and the 6 RayBio Biotin Label based Rat Antibody Array 1 Protocol total protein concentration is determined For each sample it is recommended that the concentration of the supernates and cell lysate help nomalize different cell culture supernates be determined 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 used Culture times may vary depending on your cell lines and research B Dialysis of Sample The cell culture supernates should be dialyzed with a Dialysis tube Item A before the biotin labeling procedure We recommend loading 2 5 3 0 ml cell culture supernates into a dialyzer and dialyzing with at least 2 000 ml 1X PBS buffer pH 8 at 4 C Change the 1X PBS buffer and dialyze again Allow at least 3 h for each dialysis step stir gently The sample total volume may be changed after dialysis Note Preparation of PBS pH 8 0 1 0 g KCI 40 g NaCl 1 0 g KH PO 5 75 g Na2HPO4 dissolve in 4 500 ml deionized or distilled water Adjust pH 6 0 with IM NaOH and adjust final volume to 5 000 ml with deionized or distilled water C Biotin labeling Sample Avoid contamination with any solution containing amines 1 Tris glycine as well as Azide during the biotinylation process 1 Briefly spin down Internal C
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