RayBio Biotin Label-based Antibody Array Trail Kit
Contents
1. tube pipette up and down or vortex slightly to dissolve the powder to prepare 1X Labeling Reagent Solution W 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 with gentle shaking for 30 min Gently tap the tube to mix the reaction solution every 5 min x For labeling cell culture supernates 36 ul of 1X Labeling Reagent Soluition for labeling 1 mg total protein in the cell culture supernates For labeling serum or plasma Add 22 ul of Labeling Reagent Solution into each prepared serum or plasma sample in page 8 step 1 4 Add 5 ul Stop Solution Item D into above reaction solution and immediately dialyze as directed in Step B 5 Samples should be centrifuged at 10 000 g for 5 min 4 C after Dialysis Note Samples supernates can be stored at 20 or 80 C until microarray assay D Dry the Glass Chip Open the box containing glass chip and take it out and then let it air dry for 1 hour in a clean environment before use Note Protect the chip from dust or others contaminants 9 RayBio label based Antibody Array Trail Kit 1 Protocol E Blocking and Incubation of Antibody Array 1 Add 100 ul of Blocking Buffer Item F into each well Glass Chip with Frame Item E and incubate at room temperature for 30 min to block slides Make sure there are no bubbles in the well 2 Decant Blockin2. 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 Connexin suppresses Mouse 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 17 RayBio label based Antibody Array Trail Kit 1 Protocol 6 LPS induces the interaction of a transcription factor LPS induced TNF a factor and STAT6 B with effects on multiple cytokines 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 res
3. J LJ LJ Barcode Assay Diagram 1 Cell Culture Supernates 2 Serum or Plasma ue i B B T B a H B o B Pise 6 RayBio label based Antibody Array Trail Kit 1 Protocol A Preparation of Cell Culture Supernates 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 for 10 min aliquoted and stored at 80 C until use Meanwhile the cells are also collected and the total protein concentration is determined For each sample it is recommended that the concentrations of the supernates and cell lysates help nomalize different cell culture supernates are 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 The culture time may be different and depends on your cell lines and research B Dialysis of Sample The cell culture supernates serum or plasma should be dialyzed with a Dialysis tube Item A before the b
4. RayBio Biotin Label based Antibody Array Trail Kit 1 For the Simultaneous Detection of the Expression Levels of Multiple Target Proteins in Cell Culture Supernates Serum and Plasma User Manual Glass slide based Array RayBio Biotin Label based Antibody Array Human Trail Kit 1 Cati AAH BLG TK1 detect human target proteins RayBio Biotin Label based Antibody Array Mouse Trail Kit 1 Cat AAM BLG TK2 detect mouse target proteins RayBio Biotin Label based Antibody Array Rat Trail Kit 1 Cat AAR BLG TK3 detect rat target proteins mmm mum 2 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 EL UnsodUOHO eei 232252929 55 ai PEDES bs Edel obese HOW It WODKSi s coi rris Siio o ERE EERERPRRI EIU Pad ddd II Materials Provided ccccc cece cece eens III Additional Materials Required IV Overview and General Considerations A Handling Glass Chips usuuuus B Incubation of Antibody Array Wu JOIDEOLaien dede sioe pue eM debe Ere A Preparation of Samples B Dialysis of Sample cc00 urrien C Biotin labeling Sample 006 D Dry the Glass Chip a issued etos hos tu eR d
5. as E Blocking and Incubation of Antibody Array F Fluorescence Detection esses VI Interpretation of Results cece eee VII Troubleshooting Guide cccce cece ee eee WIG Reference T 85Euosssoxezes cecsteeetecreesccedneciu nen RayBio label based Antibody Array Trail Kit 1 Protocol nn BW W WN VN tA I Introduction Recent technological advances by Raybiotech have enabled the largest commercially available antibody arrays to date With our label based antibody arrays researchers can now obtain a broad panoramic view of cytokine expression The expression levels of several hundreds of target proteins can be simultaneously detected including cytokines chemokines growth factors angiogenic factors proteases soluble receptors and other proteins in cell culture supernate and other body fluids 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 Antibody Array is to biotinylate the primary amine of the proteins in cell culture supernates serum and plasma 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 2 RayBio label based Antibody Array Trail Kit 1 Proto
6. col Here s how it works I bioti d samples dL lotin label reagent array support rd oh ee ot Vu V7 Vv biotin labeled samples incubation with labeled streptavidin signal detection II Materials Provided Upon receipt the kit should be stored at 20 C Please use within 6 months from the date of shipment After initial use the Blocking Buffer Stop Solution 20X Wash Buffer I and II Serum Buffer and Fluorescent dye Conjugated Streptavidin cy3 3 RayBio label based Antibody Array Trail Kit 1 Protocol equivalent should be stored at 4 C to avoid repeated freeze thaw cycles The Array 1 Glass Chip and internal control should be kept at 20 C Dialysis tube Item A 8 tubes Labeling Reagent Item B 2 tubes Internal control Item C 2 tubes Stop Solution Item D 1 tube RayBio Biotin label based antibody array trail kit 1 or trail kit 2 or trail kit 3 Glass Chip with Frame Item E each slide with 4 Subarrays Blocking Buffer Item F 8 ml 20X Wash Buffer I Item G 30ml 20X Wash Buffer II Item H 30ml Fluorescent dye Conjugated Streptavidin Item I cy3 equivalent 2 tubes Adhesive film Item J Serum Buffer Item K 8 ml D Tube Floating Rack 30 ml centrifuge tube III Additional Materials Required 1X PBS pH 8 0 Orbital shaker 1 ml tube 15 ml and 50 ml conical collection tube Laser scanner for fluorescence detection Aluminum foil 4 RayBio label bas
7. dequate 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 High backround 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 Avoid bubble formation during incubation Arrays are not completely covered by reagent Completely cover arrays with solution RayBio label based Antibody Array Trail Kit 1 Protocol VIII Reference List l 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 Mouse prostate cancer sera Antibody screening and identification of potential biomarkers Jeremy C Miller Heping Zhou Joshua Kwekel Robert Cavallo et al The Brian B
8. earch 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 five 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 18 RayBio label based Antibody Array Trail Kit 1 Protocol RayBiotech Inc the protein array pioneer company strives to research and develop new products to meet demands of the biomedical community RayBio s patent pending technology allows detection of over 180 cytokines chemokines and other proteins in a single experiment Our format is simple sensitive reliable and cost effective Products include Cytokine Arrays Chemokine Arrays ELISA kits Phosphotyrosine kits Recombinant Proteins Antibodies and custom services Antibody arrays 2 Cytokine antibody array Human cytokine antibody arrays Mouse cytokine antibody arrays Rat cytokine antibody arrays Pathway or disease focused antibody arrays Inflammation antibody array Angiogensis antibody array Chemokine antibody array Growth factor antibody array MMP antibody array Atherosclerosis a
9. ed Antibody Array Trail Kit 1 Protocol Distilled water IV Overview and General Considerations A Handling glass chips The microarray slides are sensitive so do not touch the array surface by tips forceps or hand Hold the slides by the edges only Handle the slides with latex free gloves Avoid breaking the glass slide A clean environment is essential Remove the final buffer by gently applying suction with a pipette to corners of each chamber see the following picture Do not touch the array only the sides Incubation of Antibody Array Completely cover the array area with sample or buffer during incubation and cover the incubation chamber with adhesive film or a plastic sheet protector to avoid drying particularly when incubation is more than 2 hours Avoid foaming during incubation steps Perform all incubation and wash steps under gentle rotation 5 RayBio label based Antibody Array Trail Kit 1 Protocol e Avoid cross contamination by preventing overflow to neighboring wells e Several incubation steps such as step 2 sample incubation in page 10 or step 7 Fluorescent dye Conjugated Streptavidin incubation in page 11 may be done at 4 C for overnight Please make sure to cover the incubation chamber tightly to prevent evaporation e Avoid array slide exposure to light since step 6 in page 11 V Protocol Layout of Array Glass Chip 4 Subarrays in one glass chip Blank LJ LJ LJ LJ L
10. g Buffer from each well make sure to remove all of buffer Add 100 ul of each sample into appropriate wells Incubate arrays with sample at room temperature with gentle shaking for 2 hours or 4 C for overnight Note a We recommended dilution of the biotin labeled cell culture supernates 2 10 fold or the biotin labeled serum plasma 40 fold with blocking buffer Make sure there are no bubbles in the wells b The dilution used depends on the abundance of target proteins The sample can be diluted further if the background or signal is too strong If the signal is too weak more sample can be used c Incubation may be done at 4 C overnight d Avoid the flow of samples into neighboring wells 3 Decant the samples from each well and wash 3 times with 200 ul of 1X Wash Buffer I Item G at room temperature with gentle shaking for 5 min per wash 4 Put the glass chip with frame into a box with Wash Buffer I 10 RayBio label based Antibody Array Trail Kit 1 Protocol cover the whole glass slide and frame with Wash Buffer I and wash at room temperature with gentle shaking for 20 min 5 Decant the Wash Buffer I from each well Put the glass chip with frame into the box with Wash Buffer II cover the whole glass slide and frame with Wash Buffer II and wash 2 times at room temperature with gentle shaking for 5 min 6 Remove all of Wash Buffer II in the well Add 100 ul of 1X Fluorescent dye Conjugated Streptavidin to eac
11. h subarray Cover the incubation chamber with Adhesive film and then cover the plate with aluminum foil to avoid exposure to light or incubate in dark room Note briefly spin down the Fluorescent dye Conjugated Streptavidin Item I before use Add 50 ul of Blocking Buffer into the tube to prepare a Streptavidin Concentrate Pipette up and down to mix gently don t store the Concentrate for next day use Add 5 ul of Streptavidin Concentrate into a tube with 500 ul of Blocking Buffer Mix gently to prepare 1X Streptavidin solution 7 Incubate at room temperature with gentle shaking for 2 hours Note Incubation may be done at 4 C for overnight 8 Decant the solution and disassemble the slide out of the incubation frame and chamber Disassemble the device by pushing clips outward from the slide Carefully remove the slide from the gasket 11 RayBio label based Antibody Array Trail Kit 1 Protocol Note Be careful not to touch the surface of the array side 9 Gently put the glass chip into a 50 ml centrifuge tube as shown in the following figure A or a plastic box with 40 ml of 1X Wash Buffer I Gently roll the tube or shake for 10 min Remove the wash buffer Repeat 2 times for a total of 3 washes A B 10 Wash the glass chip with 40 ml of 1X Wash Buffer II Repeat one time for a total of two washes for 5 min per wash 12 RayBio label based Antibody Array Trail Kit 1 Protocol 11 Finally wash the glass chip
12. iotin labeling procedure We recommend loading 200 ul cell culture supernates or 5 fold diluted serum plasma with 1X PBS pH 8 20 ul serum or 7 RayBio label based Antibody Array Trail Kit 1 Protocol plasma 80 ul 1X PBS into a dialyzer and dialyzing with at least 500 ml 1X PBS buffer pH 8 at 4 0C Change the 1X PBS buffer and dialyze again Allow at least 3 h for each dialysis step stir gently Note Preparation of 1X PBS pH 8 0 0 6 g KCI 24 g NaCl 0 6 g KH PO 3 45 g Na2HPO4 dissolve in 2500 ml deionized or distilled water Adjust pH 8 0 with IM NaOH and adjust final volume to 3000 ml with deionized or distilled water C Biotin labeling Sample Avoid contamination with any solution containing amines 1 e Tris glycine as well as Azide during the biotinylation process 1 Briefly spin down Internal Control tube Item C before use Add 100 ul 1X PBS pH 8 0 into the Internal Control tube pipette up and down to dissolve the powder a For labeling cell culture supernates add 40 ul prepared Internal Control into each tube containing 180 ul dialyzed cell culture supernates b For labeling serum or plasma add 40 ul prepared Internal Control into each tube containing 35 ul dialyzed serum or plasma sample and 155 ul Serum Buffer Item K 2 Immediately before use briefly spin down the Labeling Reagent tube Item B Add 100 ul 1X PBS into the 8 RayBio label based Antibody Array Trail Kit 1 Protocol
13. ntibody array Antibody analysis tool software ELISA Cell based phosphorylation assay Custom antibody arrays Antibody Recombinant protein Cytokine protein arrays Quantibody arrays for quantitative measurement of cytokine and other protein concentration OMONrAN NF W 19 RayBio label based Antibody Array Trail Kit 1 Protocol 10 Phosphorylation antibody arrays 11 Biotin label based antibody arrays for high density antibody arrays RayBiotech also provides excellent custom service 1 Antibody arrays 2 Protein arrays 3 Peptide synthesis 4 Production of recombinant protein and antibody 5 Peptide arrays 6 Phosphorylation arrays 7 ELISA Just simply send your samples and we will do the assay for you Technology transfer program Have you developed technologies or reagents interested to the scientific and research community RayBiotech can help you commercialize your technologies reagents and dream 20 RayBio label based Antibody Array Trail Kit 1 Protocol This product is for research use only 2004 RayBiotech Inc 21 RayBio label based Antibody Array Trail Kit 1 Protocol
14. tation and help normalize the results from different arrays being compared Antibody affinity varies significantly The fluorescence intensity detected on the array with each antibody depends on its 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 Label based Antibody Arrays Trail Kit 1 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 RayBio Biotin Label based Anitibody Array Trial Kit 1 Map Cati AAH BLG TK1 A B C D E G H l J K L M aon ona A QQ N 14 RayBio label based Antibody Array Trail Kit 1 Protocol RayBio Biotin Label based Anitibody Array Trial Kit 2 Map Cat AAM BLG TK2 B C D E A F G H l J IL 17 MCP 1 MCP 5 MIP 1a MIP 2 MIP 38 RANTES IL 17 MCP 1 MCP 5 MIP 10 MIP 2 MIP 38 RANTES OnNoaOua RWODND RayBio Biotin Label based Anitibody Array Trial Kit 3 Map Cat AAR BLG TK3 Fas Ligand GM CSF ICAM 1 Fas Ligand Fractalkine GM CSF ICAM 1 15 RayBio label based Antibody Array Trail Kit 1 Protocol VII Troubleshooting Guide Problem Cause Recommendation Weak signal Inadequate detection Check laser power and PMT parameters Ina
15. with 40 ml of deionized or distilled water for 5 min Note You may assemble the glass chip into an incubation chamber by following step if necessary You may want to practice assembling the device with a blank glass slide as shown in the figure B a Apply slide to incubation chamber barcode facing upward as in step a b Gently snap one edge of a snap on side as shown in step b c Gently press other of side against lab bench and push in direction shown in step c d Repeat with the other side F Fluorescence Detection Put the glass chip into a 50 ml centrifuge tube and dry the glass chip by centrifuge at 1 000 rpm for 3 minutes or dry the glass chip by a compressed N stream You can also let the glass chip dry completely in a clean environment protect from light Make sure the slides are absolutely dry before the scanning procedure The signals can be visualized with a laser scanner such as the Axon GenePix using the cy3 channel Note We recommend scanning the slides right after the experiment You also can store the slides at 20 C in the dark for several days If you do not have a laser scanner we can provide this service for you Just simply send your slide to us and we will take care of it 13 RayBio label based Antibody Array Trail Kit 1 Protocol VI Interpretation of Results A biotinylated protein and internal control will produce positive control signals which can be used to identify the orien
Download Pdf Manuals
Related Search