™ Exosome Antibodies, Array & ELISA Kits
Contents
1. of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBl is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2014 System Biosciences SBI All Rights Reserved Page 18 ver 6 2014 01 02 www systembio com2. sample exosomes from serum or culture media using ExoQuick or ExoQuick TC also compatible with exosome pellets from ultracentrifugation to obtain an exosome pellet We suggest when using ExoQuick start with 500 ul serum sample 300 ul PBS For culture media we suggest starting with 10 ml media and then resuspend exosome pellet with 300 ul PBS 2 Usea small volume of resuspended exosomes and perform a protein assay to determine the recovery Add 300 500 ug exosome proteins with 600 ul Exosome Lysis buffer Vortex for 15 seconds to prepare exosome protein lysate mixture In a fresh 15 mL Falcon tube combine the 600 ul exosome lysate with 9 4 mL Exosome Array Binding buffer and mix by inverting 3 times 6 Inasmall clean tray briefly wet the antibody array in 5 mL distilled water for 2 minutes at room temperature 7 Decantthe water from the membranes Pipette the 10 mL Exosome lysate binding mixture to one Exo Check antibody membrane array Place the membrane face up by positioning the membrane such that the upper left notched corner can be seen in the left hand side on top 8 Incubate the tray membrane mixture shaking at 2 8 C overnight on a shaker or a rocker e EOD Antibody array washing and signal detection 1 Decantthe Exosome lysate binding mixture from the membrane carefully 2 Add 10 mL Array wash buffer made into 1X from 20X stock and shake gently for 5 minutes at room temperature 3 Decantthe Array wash b
3. 1 000 dilution and Exosome validated secondary antibody was used at 1 20 000 dilution We recommend the SuperSignal West Femto Chemi luminescent Substrate THERMO catalog 34095 Exosome Antibody Arrays Cat EXORAY series The Exo Check antibody array has 12 pre printed spots and features 8 antibodies for known exosome markers CD63 CD81 ALIX FLOT1 ICAM1 EpCam ANXA5 and TSG101 and a GM130 cis Golgi marker to monitor any cellular contamination in your exosome isolations The array is compatible with ultracentrifugation and ExoQuick methods The positive control spot is derived from human serum exosome proteins and are printed directly onto the membrane Your exosome preparations are lysed and then incubated with the array for the pre printed antibodies to capture their respective exosome proteins The GM130 antibody spot on each array is included to help identify and cellular contamination in your exosome preparations The kits come complete with a secondary detection mixture conjugated to HRP The arrays are semi quantitative and can be used to evaluate relative abundance of certain exosome protein 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual markers from a set of given samples The orientation of the Array is identified as using the upper left hand corner that is notched l Protocol for Exo Check Antibody Arrays Sample preparation and antibody array capture 1 Precipitate
4. 1 or 25 ul Hsp70 Exosome validated secondary antibody Goat anti Rabbit HRP gt p The Exosome Antibodies are shipped in blue ice and should be stored at 4 C upon receipt Properly stored antibodies are stable for 6 months from the date received They can be place at 20 C for long term storage EXORAY Series Exo Check Exosome Antibody Arrays Antibody Membrane Array PVDF membrane 4 8 or 12 array membranes Exosome Lysis buffer Exosome Array Binding buffer Array Wash buffer Detection buffer HRP conjugated secondary mixture Disposable blue forceps to handle membrane arrays The Exosome Antibody array kits are shipped in blue ice and should be stored at_ 4 C upon receipt Properly stored antibody array kits are stable for 6 months from the date received Not supplied HRP developer solution we recommend the SuperSignal West Femto Chemi luminescent Substrate THERMO catalog 34095 Page 2 ver 6 2014 01 02 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXORAY EXOEL Series EXOEL Series ExoELISA kit Components Amount Exosome binding buffer 20 ml 20X Wash buffer 18 ml Blocking buffer 30 ml ExoELISA protein standard 400 ul Exosome specific primary antibody CD63 CD9 or CD81 2x25 ul Exosome validated secondary antibody Goat anti Rabbit HRP op ELISA substrate Super sensitive TMB 6 ml Stop buffer 6 ml 96 well ExoELISA plat
5. 1X Wash buffer 8 Dilute Exosome validated secondary antibody 1 5 000 1X blocking buffer and add 50 ul to each well and incubate at room temperature for 1 hour with shaking 9 Wash the plate 3 times for 5 minutes each with 100 ul 1X Wash buffer 10 Add 50 ul of Super sensitive TMB ELISA substrate and incubate at room temperature for 15 to 45 minutes with shaking 15 to 45 minutes substrate incubation time is optimized for the recommended exosome protein standard curve Page 12 ver 6 2014 01 02 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXORAY EXOEL Series Further optimization maybe required by the user for individual sample 11 Add 50 ul of Stop buffer to provide a fixed endpoint for the assay Note that the initial color of a positive sample is blue and the color changes to yellow when Stop buffer is added 12 absorbance E Sample Data CD63 Serum yr S ile e C104 Penn 00450 nm Quantitate results with a spectrophotometric plate reader at 450 nm CD9 Serum y 6E 11x 0 092 R 0 992 0 5E 9 1E 10 15E 10 5 0 SE 9 1E 10 1 5E 10 Exosome Particles 8 Exosorne Particles CD81 Serum ExoELISA Standards y 7E 11x 0 124 Calibrated by NanoSight R 0993 NANO 1 f E l 3 i i j SE 9 it Exosome Particles 1E 10 1 5E 10 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual IV Pro
6. 200 outside US Page 15 VI System Biosciences SBI User Manual Pegtel DM Cosmopoulos K Thorley Lawson DA van Eijndhoven MA Hopmans ES Lindenberg JL de Gruijl TD Wordinger T Middeldorp JM Functional delivery of viral miRNAs via exosomes Proc Natl Acad Sci USA 2010 Apr 6 107 14 6328 33 Mathivanan S and Simpson R J ExoCarta A compendium of exosomal proteins and RNA Proteomics 2009 21 4997 5000 Thery C Ostrowski M Segura E Membrane vesicles as conveyors of immune responses Nat Rev Immunol 2009 8 581 93 Michael A Bajracharya SD Yuen PS Zhou H Star RA Illei GG Alevizos Exosomes from human saliva as a source of microRNA biomarkers Oral Dis 2010 Jan 16 1 34 8 Luo SS Ishibashi O Ishikawa G Ishikawa T Katayama A Mishima T Takizawa T Shigihara T Goto T Izumi A Ohkuchi A Matsubara S Takeshita T Takizawa T Human villous trophoblasts express and secrete placenta specific microRNAs into maternal circulation via exosomes Biol Reprod 2009 Oct 81 4 717 29 Taylor DD Gercel Taylor C MicroRNA signatures of tumor derived exosomes as diagnostic biomarkers of ovarian cancer Gynecol Oncol 2008 Jul 110 1 13 21 Simpson RJ Lim JW Moritz RL Mathivanan S Exosomes proteomic insights and diagnostic potential Expert Rev Proteomics 2009 Jun 6 3 267 83 Review Technical Support For more information about SBI products and to download manuals in PDF for
7. C for 5 minutes Chilled on ice for 5 minutes before loading onto gel Perform standard SDS PAGE electrophoresis and Western transfer onto PVDF membrane Block with 5 dry milk in Tris Buffered Saline 0 05 Tween TBS T for 1 hour Incubate blot overnight at 4 C with Exosome specific primary antibody e g CD9 at 1 1000 dilution 5 dry milk in TBS T Wash 3X with TBS T Incubate one hour at room temperature with Exosome validated secondary antibody Goat Rabbit HRP antibody at 1 20 000 dilution 5 dry milk in TBS T Wash 3X with TBS T Incubate blot with chemi luminescence substrate and visualize on film or other imaging equipment We recommend the SuperSignal West Femto Chemi luminescent Substrate THERMO catalog 34095 1X RIPA buffer contains 25mM Tris HCl pH 7 6 150mM NaCl 1 NP 40 1 sodium deoxycholate 0 1 SDS 2X Laemmli buffer contains 4 SDS 20 glycerol 1096 2 mercaptoethanol 0 00496 bromophenol blue 0 125 M Tris HCl pH 6 8 ver 6 2014 01 02 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXORAY EXOEL Series II Serum Exosome Western Data gt ExoAbs D a D SS a O qe o 9 ov X o aS 9 kDa PI PS 100 MW e I 70 55 35 25 ExoQuick Exosome Serum Western Analysis Western blot analysis of serum exosomes using the recommended protocol 20 ug of serum exosome proteins were loaded into each lane All Exosome specific primary antibodies were used at 1
8. a SBI System Biosciences Exosome Antibodies Array amp ELISA Kits Cat s EXOAB EXORAY EXOEL User Manual See page 2 for product storage conditions A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement contained in this user manual Exosome Antibody and ELISA Kits Cat s EXOAB EXORAY EXOEL Series Contents Listof components sss 2 Exosome Antibodies L Recommended protocol sss sss 3 ll Serum exosome Western data 5 Exosome Antibody Arrays Exo Check ME incre 6 ll Example data veces erento rure pR e EE RE snes 7 ExoELISA Kits L ExoELISA principle sss 8 ll Equipment supplied byuser ss 9 lll Protocol 9 A Exosome precipitation with ExoQuick ExoQuick TC 9 B Exosome isolated with ultracentrifugation method 10 C Exosome protein standard 10 D ELISAprocedures sss 11 E Sample Data 13 IV CiRATIONS ae asii ee deana daa causes a rda ADENN SEEEN 14 V Technical references c scecececeeeeceaeeeeeeceaeeceeeaeers 15 VI Technical Support sss 16 VII Licensing and Warranty Statement ss 18 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual List of Components EXOAB Series Exosome Antibodies Componenis Amount Exosome specific primary antibody CD63 CD9 CD8
9. duct Citations Ramteke A Ting H Agarwal C Mateen S Somasagara R Hussain A Graner M Frederick B Agarwal R Deep G Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules Mol Carcinog 2013 Dec 17 doi 10 1002 mc 22124 Dubovsky JA Chappell DL Harrington BK Agrawal K Andritsos LA Flynn JM Jones JA Paulaitis ME Bolon B Johnson AJ Byrd JC Muthusamy N Lymphocyte cytosolic protein 1 is a chronic lymphocytic leukemia membrane associated antigen critical to niche homing Blood 2013 Nov 7 122 19 3308 16 Masato Mitsuhashi Dennis D Taub Dimitrios Kapogiannis Erez Eitan Linda Zukley Mark P Mattson Luigi Ferrucci Janice B Schwartz and Edward J Goetzl Aging enhances release of exosomal cytokine mRNAs by Ap1 42 stimulated macrophages FASEB J 2013 27 5141 5150 Sohel MMH Hoelker M Noferesti SS Salilew Wondim D Tholen E et al Exosomal and Non Exosomal Transport of Extra Cellular microRNAs in Follicular Fluid Implications for Bovine Oocyte Developmental Competence PLoS ONE 8 11 e78505 Alhasan A H Patel P C Choi C H J and Mirkin C A Exosome Encased Spherical Nucleic Acid Gold Nanoparticle Conjugates as Potent MicroRNA Regulation Agents Small doi 10 1002 smll 201302143 Lee HK Finniss S Cazacu S Bucris E Ziv Av A Xiang C Bobbitt K Rempel SA Hasselbach L Mikkelsen T Slavin S Brodie C Mesenchymal stem c
10. e 12x8 well strips 1 plate The ExoELISA kits are shipped in blue ice and should be stored at 4 C upon receipt Properly stored kits are stable for 6 months from the date received ExoELISA protein standards should be stored at 20 C upon receipt We recommend making single use aliquots to avoid repeated freeze thawing Exosome Antibodies Cat EXOAB series l Recommended protocol for isolating serum exosomes for Western blotting analysis 1 If frozen thaw 250 ul serum on ice Centrifuge at 1500 x g for 15 minutes to remove cells and cell debris Transfer sample supernatant to a centrifuge tube Combine 250 ul sample 63 ul ExoQuick Mix well by inversion three times Place at 4 C for at least 30 minutes Centrifuge at 1500 x g for 5 minutes Remove supernatant keep exosome pellet Centrifuge at 1500 x g for 5 minutes to remove all traces of fluid take great care not to disturb the pellet m 0 0 n 9 or Ac 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual 10 11 12 13 14 15 16 17 18 19 20 21 Page 4 Add 200 ul RIPA buffer with appropriate protease inhibitor cocktail added to exosome pellet and vortex 15 seconds Place at room temperature for 5 minutes to allow complete lysis Perform standard Bradford protein assay to determine yield Add Laemmli buffer with Beta mercaptoethanol and heat at 95
11. ells deliver synthetic microRNA mimics to glioma cells and glioma stem cells and inhibit their cell migration and self renewal Oncotarget 2013 Feb 4 2 346 61 Ariza ME Rivailler P Glaser R Chen M Williams MV Epstein Barr Virus Encoded dUTPase Containing Exosomes Modulate Innate and Adaptive Immune Responses in Human Dendritic Cells and Peripheral Blood Mononuclear Cells PLoS ONE 8 7 e69827 Chugh PE Sin S H Ozgur S Henry DH Menezes P et al Systemically Circulating Viral and Tumor Derived MicroRNAs in KSHV Associated Malignancies PLoS Pathog 9 7 e1003484 doi 10 1371 journal ppat 1003484 Bashratyan R Sheng H Regn D Rahman Mu Dai YD Insulinoma released exosomes activate autoreactive marginal zone like B cells that expand endogenously in prediabetic NOD mice Eur J Immunol 2013 Jul 2 doi 10 1002 eji 201343376 Page 14 ver 6 2014 01 02 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXORAY EXOEL Series Epple LM Griffiths SG Dechkovskaia AM Dusto NL White J Ouellette RJ Anchordoquy TJ Bemis LT Graner MW Medulloblastoma Exosome Proteomics Yield Functional Roles for Extracellular Vesicles 2012 PLoS ONE 7 7 e42064 doi 10 1371 journal pone 0042064 Singh PP Smith VL Karakousis PC Schorey JS Exosomes Isolated from Mycobacteria Infected Mice or Cultured Macrophages Can Recruit and Activate Immune Cells In Vitro and In Vivo J Immunol 2012 Jun 20 Epub ahead of print Alva
12. mat please visit our web site http www systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 265 North Whisman Road Page 16 ver 6 2014 01 02 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXORAY EXOEL Series Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com 888 266 5066 Toll Free 650 968 2200 outside US Page 17 VII System Biosciences SBI User Manual Licensing and Warranty Statement Limited Use License Use of the ExoAB antibodies Exo Check antibody Arrays and ExoELISA Kits i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product may not be resold modified for resale or used to ma
13. ndard curve B Exosomes isolated with ultracentrifugation method 1 Performed exosome isolation by the user s preferred ultra centrifugation protocol 2 Carefully remove all traces of supernatant keep exosome pellet 3 Resuspend exosome pellet in 200 ul of Exosome binding buffer and vortex 15 seconds 4 Incubate at room temperature for 10 minutes and keep on ice 5 Exosome protein is now ready for immobilization onto micro titer plate Proceed to section C Exosome protein standard curve C Exosome protein standard curve A standard curve should be prepared each time the assay is performed 1 Thaw ExoELISA protein standard on ice 2 Dilute ExoELISA protein standard by performing serial dilutions with Exosome Binding buffer in microcentrifuge tubes 3 Suggested dilutions for standard curve Page 10 ver 6 2014 01 02 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXORAY EXOEL Series Dilution ExoELISA Exosome Tube Exosomes pee protein binding standard buffer 0 0 Blank 60 1 1 35x10 1 60 pl 2 6 75x10 1 2 60 pl 60 pl 3 3 37x109 14 60 pl 60 pl 4 1 68x10 1 8 60 pl 60 pl 5 8 44x108 1 16 60 pl 60 pl 6 4 21x108 1 32 60 pl 60 ul 7 2 10x108 1 64 60 pl 60 pl 60ul Wiese I o MANO ___ NS A 60ul eg j Conc tration patda te 106 D ELISA procedures Before starting 1 The ExoELISA micro titer plate is provided in a convenient 8 well X 12 stri
14. nt LII Positive Postve control for HRP memor ExoELISA Cat EXOEL series l ExoELISA principle The ExoELISA kit is designed as a direct Enzyme Linked ImmunoSorbent Assay ELISA The lysed exosome particles and their proteins are directly immobilized onto the wells of the microtiter plate After binding the wells are coated with a block agent to prevent non specific binding of the primary detection antibody The detection primary antibody is added to the wells for binding to a specific antigen e g CD63 protein present in the exosome lysates A Horseradish Peroxidase enzyme linked secondary antibody Goat anti Rabbit is used for signal amplification and to increase assay sensitivity A colorimetric substrate Extra sensitive TMB is used for the assay read out The accumulation of the colored product is proportional to the specific antigen present in each well The results are quantitated by a microtiter plate reader at 450 nm absorbance Page 8 ver 6 2014 01 02 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXORAY EXOEL Series Equipment to be supplied by user Microtiter plate sealing film cover 37 C incubator Microtiter plate shaker Microtiter plate spectrophotometer with 450 nm absorbance capability Multichannel pipets recommended m RON Protocol A Exosomes precipitation with ExoQuick ExoQuick TC For simple and quick isolation of exosomes from serum we recommend using the Ex
15. nufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants that the Product meets the specifications described in this manual If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use
16. ny signal and this serves as a background control The GM130 control is to evaluate cellular contamination if any in your exosome preparations The data below are from 300 ug of exosome proteins isolated using ExoQuick TC from human HT1080 lung sarcoma cell line media Array format E 2 i 3 I 4 TI 5 a 6 F A Positive i GM130 i FLOT 1 ICAM ALIX CD81 B CD63 EpCAM ANXAS TSG101 Blank Positive Positive GM130 FLOTI ICAM ALIX CD81 wy gt o e CD63 EpCAM ANXAS TSG101 Blank Positive 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Information about the exosome protein targets y I Location us Gene Name Exocarta Link Comments i f Al Positive Postivecontro for HRP Sheer Aa Bak Wegative contol for bechqround No eredody loaded tex for background nal A3 LOT pulni Tumuzcesocamazra aene warmay pene ide 1211 i 4 i At ICAMI nteecellular adhewuonmeolecole https exocattaorg gene seminary gene d PODHAL AUN Le m Frog amieswed cell death AS ALIX http vescataorg gene sammary gene 142 1571 f nteracting peotein ALG 2 yrteractirsg protein 1 Hp i JGSFS immunoglobulin e cos http teocamaoea gene semmary gene 1d2911 famiy meenbe 81 coes bed LAMPS i 1 amp 2 pCam Jrpthelual cell devewen moleade 83 ANKAS Quneunas i B4 T G381 Tur wruepeballty gern 1 LL 3 B5 GM130 kieGolgi mamx protein d vYaminants prese
17. oQuick precipitation solution Catalog EXOQ5A 1 or EXOQ20A 1 and the ExoQuick TC for isolation of exosomes from tissue culture media and urine samples EXOTC10A 1 or EXOTC50A 1 1 If frozen thaw sample on ice 2 Centrifuge at 3000 x g for 15 minutes to remove cells and cell debris 3 Transfer supernatant to a sterile vessel and add the appropriate volume of ExoQuick or ExoQuick TC Incubation Bio fluid e ExoQuick ExoQuick TC time volume voulme 30 minutes Serum Overnight Ascites fluid 250 ul 63 ul Overnight Culture Media 5 ml 1ml Overnight Urine 5 ml 1ml Overnight Spinal fluid 5 ml 1ml 4 Mix well by inversion three times 5 Place at 4 C from 30 minutes to overnight according to table 6 Centrifuge at 1500 x g for 30 minutes 7 Remove supernatant keep exosome pellet 8 Centrifuge at 1500 x g for 5 minutes to remove all traces of fluid take great care not to disturb the pellet 9 Add 200 yl Exosome Binding buffer to exosome pellet and vortex 15 seconds 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual 10 Incubate at 37 C temperature for 20 minutes to liberate exosome proteins 11 Centrifuge at 1500 x g for 5 minutes to remove all residual precipitation solution 12 Transfer supernatant to new centrifuge tube on ice 13 Exosome protein is now ready for immobilization onto micro titer plate Proceed to section C Exosome protein sta
18. p format We recommend using at least one strip for the standard curve and additional strips depending on the number of samples tested Unused 8 well strips can be removed and stored at room temperature for later use 2 Make sure to warm the Super sensitive TMB ELISA substrate to room temperature before adding to the ELISA plate wells in step 10 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual 3 Dilute stock 20X Washing buffer into 1X working Wash buffer with purified water each 8 well strip requires approximately 10 ml of 1X Washing solution ELISA assay 1 Add 50 ul of prepared protein standards and exosome protein sample to the appropriate well of the micro titer plate 2 Cover plate with sealing film cover 3 Incubate the plate at 37 C from 2 hours to overnight recommended 4 After incubation step invert the plate to empty all contents 5 Wash the plate 3 times for 5 minutes each with 100 ul 1X Wash buffer A micro titer plate shaker is recommend for all subsequent the washing and incubation steps Residual liquid should be removed by hard tapping the plate on fresh paper towels while taking care not to let the wells dry out completely 6 Dilute Exosome specific primary antibody CD63 CD9 or CD81 1 100 in 1X blocking buffer and add 50 ul of to each well and incubate at room temperature for 1 hour with shaking 7 Wash the plate 3 times for 5 minutes each with 100 ul
19. rez ML Khosroheidari M Kanchi Ravi R Distefano JK Comparison of protein microRNA and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers Kidney Int 2012 Jul 11 doi 10 1038 ki 2012 256 V Technical References Adachi T Nakanishi M Otsuka Y Nishimura K Hirokawa G Goto Y Nonogi H Iwai N Plasma microRNA 499 as a biomarker of acute myocardial infarction Clin Chem 2010 Jul 56 7 1183 5 De Smaele E Ferretti E Gulino A MicroRNAs as biomarkers for CNS cancer and other disorders Brain Res 2010 Jun 18 1338 100 11 Mitchell PS Parkin RK Kroh EM Fritz BR Wyman SK Pogosova Agadjanyan EL Peterson A Noteboom J O Briant KC Allen A Lin DW Urban N Drescher CW Knudsen BS Stirewalt DL Gentleman R Vessella RL Nelson PS Martin DB Tewari M Circulating microRNAs as stable blood based markers for cancer detection Proc Natl Acad Sci US A 2008 Jul 29 105 30 10513 8 Laterza OF Lim L Garrett Engele PW Vlasakova K Muniappa N Tanaka WK Johnson JM Sina JF Fare TL Sistare FD Glaab WE Plasma MicroRNAs as sensitive and specific biomarkers of tissue injury Clin Chem 2009 Nov 55 11 1977 83 Valadi H Ekstr m K Bossios A Sj strand M Lee JJ L tvall JO Exosome mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells Nat Cell Biol 2007 Jun 9 6 654 9 888 266 5066 Toll Free 650 968 2
20. uffer 4 Add 10 mL Detection buffer to Array membrane and gently shake at room temperature for 2 hours 5 Prepare the Developer mixture not included about 5 minutes before the end of Step 5 by combining 1 mL solution A with 1 mL solution B together We recommend the SuperSignal West Femto Chemi luminescent Substrate THERMO catalog 34095 6 Decantthe Detection buffer 7 Add 10 mL Array wash buffer made into 1X from 20X stock and shake gently for 5 minutes at room temperature 8 Decantthe Array wash buffer repate twice for a total of three washes Page 6 ver 6 2014 01 02 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXORAY EXOEL Series 9 Addthe 2 mL Developer solution mixture from Step 5 directly onto the Array membrane so that it is completely covered 10 Incubate at room temperature for 2 minutes 11 Decant the developer solution and image the Array signals a Ifyou use a Bio Rad ChemiDoc XRS Imager System or similar expose 4 10 seconds b Expose Array membrane to Kodak XR film for 1 3 minutes and develop in a film developer machine ll Sample Data for Exo Check Antibody Arrays What to expect The positive control signals should provide a bright signal and this will indicate that all of the detection reagents are working properly The various exosome antibody spots will provide signals of varying degree depending upon the source of the isolated exosomes The Blank spot should not have a
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