Exosome Antibodies & ELISA Kits
Contents
1. Centrifuge at 1500 x g for 5 minutes to remove all traces of fluid take great care not to disturb the pellet Add 200 yl 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 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 596 dry milk in Tris Buffered Saline 0 0596 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 596 dry milk in TBS T Wash 3X with TBS T Incubate blot with chemi luminescence substrate and visualize on film or other imaging equipment 1X RIPA buffer contains 25mM Tris HCl pH 7 6 150mM NaCl 196 NP 40 196 sodium deoxycholate 0 1 SDS 2X Laemmli buffer contains 4 SDS 20 glycerol 10 2 mercaptoethanol 0 00496 bromphenol blue 0 125 M Tris HCI pH 6 8 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual Il Serum Exosome Western2. System Biosciences Exosome Antibodies amp ELISA Kits Cat s EXOABxxx EXOELxxx 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 ver 1 2011 08 16 contained in this user manual Exosome Antibody and ELISA Kits Cat s EXOAB EXOEL Series Contents Listofcomponents sss 2 Exosome Antibodies sss 3 1 Recommended protocol ss 3 ll Serum exosome Westerndata f 4 ExoELISAkit 5 I ExoELISA principle sss 5 ll Equipment supplied by user d 5 Ill Protocol 5 VI VII A Exosome precipitation with ExoQuick ExoQuick TC 5 B Exosome isolated with ultracentrifugation method 6 C Exosome protein standard enn 7 D ELISA procedures sss uL E Sample Data 9 CITATIONS e Sisan 9 Technical references cesse eee enne nnn 9 Technical Support 10 Licensing and Warranty Statement 11 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual List of Components EXOAB Series Exosome Antibodies Components Amount Exosome specific primary antibody CD63 CD9 CD81 or 25ul Hsp70 Exosome validated secondary antibody Goat anti Rabbit Sul HRP The Exosome Antibodies are shipped in blue ice and sho
3. Data N Sy ExoAbs N of oo g ge E 9 qw ow A N a 100 MW S oC oc Ss 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 antibody were used at 1 1 000 dilution and Exosome validated secondary antibody were used at 1 20 000 dilution ExoELISA Cat EXOEL series aaron gt ExoELISA principle The ExoELISA kit is designed as a direct Enzyme Linked ImmunoSorbent Assay ELISA The exosome particles and their proteins are directly immobilized onto the wells of the microtiter plate After binding wells are coated with a block agent to prevent non specific binding of the detection antibody The detection primary antibody is added to the wells for binding to specific antigen e g CD63 protein on the exosomes 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 Equipment to be supplied by user Microtiter plate sealing film cover 37 C incubator Microtiter plate shaker Microtiter plate spectrophotometer with 450 n
4. solution 12 Transfer supernatant to new centrifuge tube on ice 13 Exosome protein is now ready for immobilization onto micro titer plate 00 N O owe Proceed to section C Exosome protein standard curve B Exosome isolated with ultracentrifugation method Performed exosome isolation by the user s preferred ultra centrifugation protocol Carefully remove all traces of supernatant keep exosome pellet Resuspend exosome pellet in 200 ul of Exosome binding buffer and vortex 15 seconds Incubate at room temperature for 10 minutes and keep on ice Exosome protein is now ready for immobilization onto micro titer plate OV POD Proceed to section C Exosome protein standard curve 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual 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 Exosomes Dilution factor ExoELISA protein standard Exosome binging buffer 0 0 Blank 60 1 1 35x10 1 60 ul F 2 6 75x10 1 2 60 ul 60 ul c3 3 3 37x10 14 60 ul 60 ul c 4 1 68x10 1 8 60 ul 60 ul 5 8 44x108 1 16 60 ul 60 ul c3 6 4 21x108 1 32 60 ul 60 ul 7 2 10x108 1 64 60 ul 60 ul c3 60ul ExoELISA
5. 9928 os 0a 02 o 5409 1E 10 Exosome Particles Page 8 1 1 5E 10 o o SE 09 1Et10 1 5E 10 Exosome Particles TMB Substrate vis gt PB HRP V Secondary Ab CD63 CD9 or CD81 Ab Exosomes directly adsorbed to ELISA Well ver 1 2011 12 12 www systembio com IV V Exosome Antibody and ELISA Kits Cat s EXOAB EXOEL Series Citations As featured in Exosome Isolation for Proteomic Analyses and RNA Profiling Douglas D Taylor Wolfgang Zacharias and Cicek Gercel Taylor Serum Plasma Proteomics Methods in Molecular Biology 2011 Volume 728 Part 4 235 246 PDF Tae Hoon Lee Esterina D Asti Nathalie Magnus Khalid Al Nedawi Brian Meehan and Janusz Rak Review Microvesicles as mediators of intercellular communication in cancer the emerging science of cellular debris Seminars in Immunopathology DOI 10 1007 s00281 01 1 0250 3 PDF 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 Gentle
6. Standards 60ul Calibrated by NanoSight A 60ul E Eu H Wa xc i B cou 1 ES 60ul 3 b B 0 H Il 3 B P 4 B i Jj js gt 6 lj JE m m NOW ND EE WR OT 7 Particle Size nm Page 6 ver 1 2011 12 12 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXOEL Series D ELISA procedures Before starting 1 The ExoELISA micro titer plate is provided in a convenient 12 well X 8 strip 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 4 on van 5 AAAS KON ve AA 0S 0790 SRI E gt aw F RORHCHUOIC UTC C 2 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 Add 50 ul of prepared protein standards and exosome protein sample to the appropriate well of the micro titer plate Cover plate with sealing film cover Incubate the plate at 37 C from 2 hours to overnight recommended After incubation step invert the plate to empty all contents 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 no
7. ations 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 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 SBI 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 2011 System Biosciences SBI All Rights Reserved 888 266 5066 Toll Free 650 968 2200 outside US Page 11
8. dar 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 manufacture 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 specific
9. m absorbance capability Multichannel pipets recommended Page 4 ver 1 2011 12 12 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXOEL Series ll Protocol A Exosome precipitation with ExoQuick ExoQuick TC For simple and quick isolation of exosomes from serum we recommend using the ExoQuick 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 time Bio fluid Sample volume ExoQuick volume ExoQuick TC voulme 30 minutes Serum 250 ul 63 ul Overnight Ascites fluid 250 ul 63 ul Overnight Culture Media 5 ml 1 ml Overnight Urine 5ml 1 ml Overnight Spinal fluid 5 ml 1 ml Mix well by inversion three times Place at 4 C from 30 minutes to overnight according to table Centrifuge at 1500 x g for 30 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 9 Add 200 ul Exosome Binding buffer to exosome pellet and vortex 15 seconds 10 Incubate at 37 C temperature for 20 minutes 11 Centrifuge at 1500 x g for 5 minutes to remove all residual precipitation
10. man R Vessella RL Nelson PS Martin DB Tewari M Circulating microRNAs as stable blood based markers for cancer detection Proc Natl Acad Sci U S 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 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 expres
11. s 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 Ru Lim JW Moritz RL Mathivanan S Exosomes proteomic insights and diagnostic potential Expert Rev Proteomics 2009 Jun 6 3 267 83 Review 888 266 5066 Toll Free 650 968 2200 outside US Page 9 Vi System Biosciences SBI User Manual Technical Support For more information about SBI products and to download manuals in PDF format 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 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 Page 10 ver 1 2011 12 12 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXOEL Series VII Licensing and Warranty Statement Limited Use License Use of the ExoAB antibodies 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 calen
12. t 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 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 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 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 Quantitate results with a spectrophotometric plate reader at 450 nm absorbance Ol ONO 888 266 5066 Toll Free 650 968 2200 outside US Page 7 OD450 nm OD450nm System Biosciences SBI Sample Data User Manual CD63 Serum CD9 Serum a y 5E 11x 0 1165 y 6E 11x 0 0916 R 0 9711 R 0 9919 12 0 T T OD450nm o2 56409 1E 10 1 5E 10 Exosome Particles CD81 Serum y 7E 11x 0 1237 R 0
13. uld 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 EXOEL Series Exosome binding buffer 20 ml 20X Wash buffer 18 ml Blocking buffer 30 ml Exosome specific primary antibody CD63 CD9 or CD81 2x25 ul Exosome validated secondary antibody Goat anti Rabbit HRP 10 ul ELISA substrate Super sensitive TMB 6 ml Stop buffer 6 ml 96 well ExoELISA plate 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 Page 2 ver 1 2011 12 12 www systembio com Exosome Antibody and ELISA Kits Cat s EXOAB EXOEL Series Exosome Antibodies Cat EXOAB series I Recommended protocol for isolating serum exosomes for Western blotting analysis 00 74 9 Or Eco 18 19 20 21 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 pl 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
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