XPEP Exosome Mass Spec Kit
Contents
1. SSBI System Biosciences XPEP Exosome Mass Spec Kit Cat XPEP100A 1 User Manual Store Kits at 20 C upon receipt 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 XPEP Exosome Mass Spec Kits Cat XPEP100A 1 Contents Le MNIROGUCH OM rM 1 A Exosome OVOIVIeW eei e ec dte 1 B The XPEP kits for exosome proteomic analysis 2 Gi APEPITIOGSdI CS ciue teeth i ee tette nad 2 D Sample XPEP exosome data cccccseeseeecesesseeeeeseeeeeeeeeeens 8 E Related Products and Services sueeueeessssse 14 F Additional Materials Required eeeeseeeessssss 17 G Shipping and Storage Conditions for Kits 17 ls Referentes ovas tenia oci tuse ta vvsnisv buius aui op VR bu ORUM NN 18 Il Technical SUDDOELU d peque sop e e ioe ue Dance eod 19 VII Licensing and Warranty information 19 l Introduction A Exosome Overview Exosomes are 60 180 nm membrane vesicles secreted by most cell types in vivo and in vitro These microvesicles are produced by the inward budding of multivesicular bodies MVBs and are released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane Exosomes are extracellular nanoshu2. Page 20 ver 1 061114 www systembio com XPEP Exosome Mass Spec Kits Cat XPEP100A 1 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 2014 System Biosciences SBI All Rights Reserved 888 266 5066 Toll Free 650 968 2200 outside US Page 21
3. 9606 Gene Symbol KRT14 Keratin type cytoskeletal 14 2 kDa ideroflexin 1 OS Homo sapiens GN SFXN1 PE 1 SV 4 36 kDa rohibitin OS Homo sapiens GN PHB PE 1 SV 1 lsp P35232 PHB_ HUMAN 43 Heat shock 70 kDa protein 1A 1B OS Homo sapiens GN HSPA1A P wIeoIApmI Ix alo 5 g9 oa Q9 o O Slo fed C4 zi O N O 2 T o 3 o uo CD oz D Vn e Z D gt O O ps u 9 m pay nN lt p Co N a lt a D be v oF D ep lt Qa D Ww Tb o Wn Tb D me D EL D lt e o oa D e D 4 D e n I o 3 o 4 ES 9 D ES Wn O 4 Ke g BK e a O Ww zs I G D gt Z Ww oo Cc r O N UI gt Ui CU m m e C lt N Heat shock protein HSP 90 beta OS Homo sapiens GN HSP90AB1 0 kDa heat shock protein mitochondrial OS Homo sapiens GN H sp P10809 CH60 HUMAN ax_Id 9606 Gene_Symbol KRT9 Keratin type cytoskeletal 9 2 kDa gt Oo TU gt 4 uv ct A fed 2 v o eo fe n D N O Nn L o 3 o n ie 9 D gt n e z Oo p oo ANXAS PE 1 SV 2 6 kDa D81 antigen OS Homo sapiens GN CD81 PE 1 SV 1 ysosome associated membrane glycoprotein 1 OS Homo sapien sp P11279 LAMP1 HUMAN D9 antigen OS Homo sapiens GN CD9 PE 1 SV 4 Heat shock protein HSP 90 alpha OS Homo sapiens GN HSP90AA 58 Poly rC binding protein 2 OS Homo sapiens GN PCBP2 PE 1SV Dolichyl diphosphooligosaccharide protein glycosyltransferase ssp PO4843 RPN1 H
4. research 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 credit 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 credit 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
5. spectrum details and counts Identify proteins intuitively and confidently VNNN Page 6 ver 1 061114 www systembio com XPEP Exosome Mass Spec Kits Cat XPEP100A 1 gt Create comprehensive lists of target proteins gt Classify proteins by molecular function or organelle gt Harness high through put batch processing Scaffold software download link htto www proteomesoftware com products free viewer Scaffold software tutorials htto www proteomesoftware com products tutorial videos Tli Load Data imil JN y Aaa Statistics Isolating Exosome using ExoQuick ExoQuick TC and Exo Flow Immunocapture kits Description Size Catalog ExoQuick Serum exosomes 75 rxns EXOQ5A 1 75 rxns EXOQ5TM 1 100 rxns TMEXO 1 300 rxns EXOQ20A 1 10 rxns EXOTC10A 1 50 rxn EXOTC50A 1 Exosome isolation protocols using ExoQuick reagents 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Combine your biofluid sample containing exosomes with ExoQuick or ExoQuick TC using the guidelines shown in the Table below Mix the ExoQuick precipitation reagent with the biofluid sample by inversion and place at 4 C for 30 minutes to overnight then recover the exosomes in a pellet with a low speed spin Please refer to the ExoQuick or ExoQuick TC User manuals for more details Recommended amounts of exosomes provided in Table Resuspend Volume to exosome use in pellet
6. with SBl s ExoQuick TC cat EXOTC10A 1 NOTE ExoQuick and ExoQuick TC for exosome isolation purposes are not provided in the XPEP kits and can be purchased separately The following ExoQuick products are recommended for exosome concentration prior to Exo Flow purification The XPEP kits come complete with 10 kD cut off spin columns for Mass Spec sample preparation The typical column and collection tube use is outlined in the figure below Recover Collect 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual XPEP Shave surface protein analysis protocol a To the exosome pellet or liquid suspension add 400 ul of Shaving buffer and mix gently by inversion 3 times Exosome are now ready for trypsin shaving of surface proteins proceed to Reduction Alkylation and Digestion steps Exosome surface protein shaving n Shaved exosome Intact exosomes Shaved exosomes peptide library XPEP Complete protein analysis protocol a To the exosome pellet or liquid suspension and add 0 5 ml of RIPA lysis buffer and vortex for vigorously for 10 seconds The sample is then heated for 15 minutes at 100 C Allow the sample cool down to room temperature for 5 minutes Transfer the sample Fill into a 10 kD spin column and centrifuge it for 10 minutes at highest speed Discard the flow through lysis solution keep the spin column Spin Recover the solubilized exosome protei
7. Exo Flow Urine 2 ml 500 uL PBS 100 uL rxn Spinal fluid 2 mi 500 uL PBS 100 pL rxn Culture media 2 ml 500 uL PBS 100 uL rxn Sample ExoQuick Biofluid volume TC volume Resuspend Volume to ExoQuick exosome use in Exo pellet Flow 250 uL SORRE 100 pLirxn 250 uL SORRE 100 pLirxn Ascites fluid 500 uL 120 pL Bo 100 pL rxn Amount of exosomes to use The number of exosomes in a given biofluid will vary depending upon the sample itself There are abundant levels of exosome in serum less in cell culture medium and urine Use the guidelines in the Tables above as a starting point Sample Bioflui ofluid volume D Sample XPEP exosome data Exosomes were first isolated from the tissue culture medium from HEK293 cells grown in Exo FBS exosome depleted media supplement with standard DMEM The cells were grown to 80 90 confluency in a 10 cm cell culture dish The secreted Page 8 ver 1 061114 www systembio com XPEP Exosome Mass Spec Kits Cat XPEP100A 1 exosomes were isolated as stated in the protocol for ExoQuick TC above The exosome pellet was processed using the XPEP complete or XPEP Shaving procedures Sample XPEP Shaved exosome NanoSight data 4 E 07 C MOM The shaved exosomes 91nm 2 01e009 particles mi TeMain intact but lose n their surface protein PlusTryp 37 C 4h coats and diameters 4 E 07 2 69e 009 icles ml M atu p
8. UMAN 69kDa 9 Annexin A2 0S Homo sapiens GN ANXA2PE 1SV 2 sp PO7355 ANXA2 HUMAN 39kKDa 9 N OILI 5 5 D x 3 gt U O n E o 3 o Uu CD D D 5 n e Z E Related Products and Services SBI offers a number of exosome research products Review them here http www systembio com exosomes Isolate Exosomes with ExoQuick and ExoQuick TC Serum Plasma MSCE One step Exosome Isolation for Serum and Media MPTE Plasma Tumor Ascites Fluid Follicular fluid Y Tissue Culture Media Urine Spinal fluid Page 14 ver 1 061114 www systembio com XPEP Exosome Mass Spec Kits Cat XPEP100A 1 Purified human cancer exosomes and and mouse dexosomes Use for RNA Protein analysis calibration standards and engineering cargo for delivery to target cells All exosomes are characterized by NanoSight for size intactness and concentration as well as tested to be CD63 positive by Western blot analysis The purified exosomes are provided frozen with gt 1x10 6 exosomes 50 ug protein Exosome Cargo Labeling Fluorescently label exosome cargo Label endogenous exosome RNAs Red and internal exosome Proteins Green to monitor exosome cargo delivery to cells in real time Rab5b Beads Exo FITC PLUS Exosomes Immunopurify Exosomes and use with FACS Selectively capture distinct subpopulations of intact exosomes based on a particular surface marker and sort b
9. alytical column at 350nL min using a 2hr reverse phase gradient both columns were packed with Jupiter Proteo resin Phenomenex The injection volume was 30uL The mass spectrometer was operated in datadependent mode with the Orbitrap operating at 60 000 FWHM and 17 500 FWHM for MS and MS MS respectively The fifteen most abundant ions were selected for MS MS and data analyzed using MASCOT databases and Scaffold software The software screenshot below and excel table below that image shows some example serum exosome protein MS MS data with common exosome proteins highlighted in yellow 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual Identified Proteins Accession Number_ mw to complete Tax 1d 9606 Gene_Symbol KRT6A Keratin type II cytoskeletalGA IPLCON 00300725 7 SWISS PROT GOkDa 8 Tax d 9606 Gene Symbol KRTS Keratin type ll cytoskeletal 5 IPLCON 00009867 3 SWISS PROT 2kDa 8 Prohibitin 2 OS Homo sapiens GN PHB2 PE 1 SV 2 eratin type Il cytoskeletal 2 epidermal OS Homo sapiens GN KR sp P35908 K22E HUMAN 5 kDa 21 Homossp P21796 VDACL HUMAN 31kDa sp P 3261 ACTG HUMAN sp P63261 ACTG HUMAN D D 2 Qo D 2 p w E o 2 Uo uk D o et lt D o 2 w 2 2 E o A o ot T 2 j O Nn m o 3 o Un wn o TU N e N Ko AeA lt J gt O me x c lt gt Z 92 pa ax Id
10. arties condense into a more U homogeneous 3 E407 population B U 26407 No Digest gu 113nm 2 2 E407 m x e UJ 1 E 07 u is 5 E406 E P p 0 00 0 100 200 300 400 so Size nm 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual The XPEP Shaved exosomes are intact but lose their surface CD63 protein marker as measured by CD63 antibody bead capture and Exo Flow FACs analysis SBI cat EXOFLOW300A 1 kit Sample XPEP Shaved exosome FACs data CD63 Beads Exo FITC CD63 Beads Exo FITC NO Exosomes PLUS Exosomes FSC H 10 10 107 10 10 10 10 10 10 10 10 10 CD63 Beads Exo FITC Shaved Exosomes 250K 7 No Exosomes 7 Shaved Exosomes Intact Exosomes 200K Count 0 2 3 E 5 10 10 10 10 10 10 Page 10 ver 1 061114 www systembio com XPEP Exosome Mass Spec Kits Cat XPEP100A 1 Sample XPEP SDS PAGE data XPEP Mass Spec protein libraries prepared by either the Shave or Complete protocols Approximately 10 ug of the peptide libraries were separated on a 4 20 gradient SDS PAGE gel and stained with Imperial blue Thermo Fisher to visualize the library peptide size distributions The peptide library fragments generated are of the expected 2 10kD fragment range and optimal for Mass Spec analysis The XPEP Shaved peptide libraries are typically 5 fold less than the libraries made using the XPEP Complete pro
11. eb site http www systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 265 North Whisman Rd SBI Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mails General Information info systembio com Technical Support tech systembio com Ordering Information orders sysitembio com Vil Licensing and Warranty information Limited Use License Use of the XPEP Kits 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 888 266 5066 Toll Free 650 968 2200 outside US Page 19 System Biosciences SBI User Manual e 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 e The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI e This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic
12. menex The injection volume is 30 uL The Mass spectrometer is operated in data dependent mode with the Orbitrap operating at 60 000 FWHM and 17 500 FWHM for MS and MS MS respectively The fifteen most abundant ions were selected for MS MS Example data processing protocol 1 Data are searched using a local copy of Mascot with the following parameters 2 Enzyme Trypsin P 3 Database Swissprot Human concatenated forward and reverse plus common contaminants Fixed modifications Carbamidomethyl C Variable modifications Oxidation M Pyro Glu N term Q Deamidation N Q Mass values Monoisotopic Peptide Mass Tolerance 10 ppm Fragment Mass Tolerance 0 02 Da Max Missed Cleavages 2 0 Mascot DAT files were parsed into the Scaffold software for validation filtering and to create a nonredundant list per sample Data are filtered using at 1 protein and peptide FDR and requiring at least two unique peptides per protein A Scaffold file is generated for the study and contains all search results coverage maps peptide lists and product ion data Les Acetyl N term PL Viewing XPEP mass spec data results We recommend using the free Scaffold software to open view and analyze the Scaffold file produced from the Mass spec instrument Scaffold allows you to visualize and validate complex MS MS proteomics experiments Compare samples to identify biological relevance Identify regulated isoforms and protein PTMs Drill down into
13. n mixture by inverting the 10 kD column and inserting it into a fresh collection tube Spin the inverted column for 2 minutes at high speed The recovered volume should be about 100 200 ul retain the spin column and collection tube for the next steps Recover Place the 10 kD column in the collection tube upright and transfer the 200 ul protein mixture to the spin column on top and add 100 ul of washing buffer Pipet up and down a few times to mix Centrifuge the spin column at high speed for 5 minutes discard flow through Buffer exchange the exosome protein mixture by adding 100 ul Digestion buffer to the spin column pipet up and down 3 times to mix This removes the urea from the mixture Page 4 ver 1 061114 www systembio com XPEP Exosome Mass Spec Kits Cat XPEP100A 1 Centrifuge the column for 10 minutes at highest speed Discard the flow through Repeat steps j k one more time for a total of 2 exchanges Recover the exosome protein mixture by inverting the 10 kD column and inserting it into a fresh collection tube Spin the inverted column for 2 minutes at high speed The recovered volume should be about 200 ul Reduction Alkylation and Digestion a b C To the 200 ul exosome protein solution in the collection tube add 1 5 ul the Reduction buffer 1a Reduce the protein solution for 15 minutes at 60 C Allow the solution to cool down at room temperature for 5 minutes Add 100 ul of Reduction buffer 1b
14. oon S Kim HC Baek JH Park HS Lim HJ Chung H Exosomal Proteins in the Aqueous Humor as Novel Biomarkers in Patients with Neovascular Age related Macular Degeneration J Proteome Res 2014 Jan 8 Epub ahead of print Mayne J Starr AE Ning Z Chen H Chiang CK Figeys D Fine Tuning of Proteomic Technologies to Improve Biological Findings Advancements in 2011 2013 Anal Chem 2013 Nov 5 Epub ahead of print Dear JW Street JM Bailey MA Urinary exosomes A reservoir for biomarker discovery and potential mediators of intra renal signaling Proteomics 2012 Nov 6 doi 10 1002 pmic 201200285 Griffin NM Schnitzer JE Overcoming key technological challenges in using mass spectrometry for mapping cell surfaces in tissues Mol Cell Proteomics 2011 Feb 10 2 R110 000935 He Z De Buck J Cell wall proteome analysis of Mycobacterium smegmatis strain MC2 155 BMC Microbiol 2010 Apr 22 10 121 doi 10 1186 1471 2180 10 121 Dreisbach A van der Kooi Pol MM Otto A Gronau K Bonarius HP Westra H Groen H Becher D Hecker M van Dijl JM Surface shaving as a versatile tool to profile global interactions between human serum proteins and the Staphylococcus aureus cell surface Proteomics 2011 Jul 11 14 2921 30 Page 18 ver 1 061114 www systembio com XPEP Exosome Mass Spec Kits Cat XPEP100A 1 Ill Technical Support For more information about SBI products and to download manuals in PDF format please visit our w
15. s and covert to cDNA for microRNA qPCR arrays or amplify exoRNAs for microarrays analysis 200 b XPEP Exosome Mass Spec Kits Cat XPEP100A 1 Discover Novel Exosome RNA Biomarkers Put your Data into Context with Next Gen Sequencing Complete exosome RNA Next Gen sequence Weary analytics solution for researchers interested in 6 identifying novel exosome associated RNA i biomarkers Abundance RNA type expression EN GS heatmaps and genomic mapping all included in service F Additional Materials Required 1 ExoQuick and or ExoQuick TC to isolate exosomes prior to making peptide libraries 2 Standard 1x PBS 3 Sterile 1 5 mL sample tubes 4 Standard bench top microfuge G Shipping and Storage Conditions for Kits The XPEP kits are shipped on dry ice and should be stored at 20 C Avoid freeze thawing the reagents Shelf life of the product is 1 year after receipt if stored in 20 C 888 266 5066 Toll Free 650 968 2200 outside US Page 17 System Biosciences SBI User Manual ll References Kadiu Narayanasamy P Dash PK Zhang W Gendelman HE Biochemical and Biologic Characterization of Exosomes and Microvesicles as Facilitators of HIV 1 Infection in Macrophages J Immunol 2012 Jun 18 Epub ahead of print Drake RH Kislinger T The proteomics of prostate cancer exosomes Expert Rev Proteomics 2014 Feb 25 Epub ahead of print Kang GY Bang JY Choi AJ Yoon J Lee WC Choi S Y
16. stion Buffer 5 ml Trypsin digestion mixture 25 ul 10 kD spin columns 25 columns Collection tubes 50 tubes OF oi oo IS cedi tO The protocol begins with either exosomes in a pellet form or in a concentrated solution The minimum input amount of exosomes as measured by protein concentration is 500 ng to 1 ug This equates to about 2 x 10 9 exosomes as starting input material All spins take place in a standard 1 5 ml rotor in a microfuge and high speed can be at 13 000 or 10 000 rpm Serum exosomes tip Use ExoQuick precipitation twice on a serum sample to remove some co purifying serum proteins To do this take 250 ul serum add 60 ul ExoQuick and incubate at 5 C Page 2 ver 1 061114 www systembio com XPEP Exosome Mass Spec Kits Cat XPEP100A 1 for 30 minutes Spin the tube for 3 minutes at highest speed Discard supernatant Resuspend the exosome pellet in 250 ul 1x PBS and add 60 ul ExoQuick Incubate at 5 C for 30 minutes and spin for 3 minutes at high speed to pellet the exosomes These are now ready for MS analysis If you have plasma samples please defibrinate using SBI s cat TMEXO 1 kit Media Urine CSF exosomes tip For studying exosomes in media from cells in culture you should grow your cells in the absence of bovine FBS SBI offers bovine exosome depleted FBS for this purpose cat EXO FBS 50A 1 Urine and CSF samples should be pre spun at 3 000 xg to pellet cellular debris prior to exosome isolation
17. to the solution and store the solution in dark cover with foil or place in a drawer at room temperature for 30 minutes Pre warm the Trypsin solution at room temperature during this step for 15 minutes Add 2 5 ul of the pre warmed Trypsin solution to the protein mixture and incubate at 37 C for 4 hours After 4 hours immediately store the reaction at 20 C to stop the reaction This is a convenient stopping point overnight Peptide library recovery a b Thaw exosome protein peptide mixture at room temperature for 5 minutes Transfer the solution to a fresh 10 kD spin column with a fresh collection tube and centrifuge the solution for 10 minutes at high speed Collect The exosome peptide library in now in the collection tube should be about 50 100 ul in volume with approximately 1 5 ug protein concentration XPEP Complete or 0 2 1 ug protein concentration XPEP Shave This peptide mixture is ready to load on most Mass spectrometers directly 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual Example Mass Spec analysis conditions Each exosome peptide library sample can be analyzed by Nano LC MS MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive Peptide mixtures are loaded on a trapping column and eluted over a 75 um analytical column at 350 nL min using a 2 hour reverse phase gradient both columns are packed with Jupiter Proteo resin Pheno
18. tocol The molecular weight marker is from Bio Rad Precision Plus Protein Dual Xtra Standards catalog 161 0377 XPEP Mass Spec Libraries 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual What to expect The protein content of exosomes can vary greatly depending upon the cell source from which it originated Some common surface and internal proteins can be observed in typical exosomes and are identified in Mass Spec data Some common proteins to look for include HSP Heat Shock proteins GAPDH Keratins Tubulin Actin Vimentin Fibulin Fibronectin Annexins Flotillins Galectin and a Enolase Cytoskeletal proteins HCV glycoproteins EBV gp350 Tetraspanins AA Immuno regulator molecules Signal transduction Membrane trafficking Lipid rafts Image adapted from David G Meckes Jr and Nancy RHaab Traub Microvesicles and Viral Infection J Virol December 2011 vol 85 no 24 12844 12854 Page 12 ver 1 061114 www systembio com XPEP Exosome Mass Spec Kits Cat XPEP100A 1 Sample serum exosome MS MS data Exosomes were isolated from 500ul control human serum using ExoQuick The exosome pellet was processed using the XPEP complete procedure The peptide libraries were then analyzed by nano LC MS MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive Peptides were loaded on a trapping column and eluted over a 75um an
19. ttle organelles that facilitate communication between cells and organs Exosomes are found in blood urine amniotic fluid breast milk malignant ascites fluids and contain distinct subsets of RNAs and proteins depending upon the cell type from which they are secreted making them useful for biomarker discovery SBI has engineered tools and provides 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual services for exosome proteomic Mass spec analysis and next generation sequencing of exosome RNA to accelerate the study of exosomes exosome protein and exosome biomarkers B The XPEP kits for exosome proteomic analysis The XPEP kits allow researchers to routinely generate high quality exosome peptide libraries for Mass spec analysis The kits work with exosomes isolated using ultracentrifugation as well as using ExoQuick serum plasma ascites samples or ExoQuick TC cell media urine spinal fluid or immunopurify specific exosome subpopulations using SBl s Exo Flow IP kits The kit comes complete to create either exosome surface protein shaving peptide libraries or complete exosome peptide library preparations C XPEP Procedures Materials provided 1 Complete exosome Lysis buffer 5 ml Shaving buffer with exosome stabilization solution 5 ml Washing Buffer 3 ml Reduction Buffer 1a 20 ul Reduction Buffer 1b add 1 ml water to stock tube before use stable for 2 weeks Dige
20. y FACS Flow Exometry Choose from the following tetraspanin annexin adhesion fusion and immune presentation l 0 Bead targets or customize your own capture system Singlets e CD9 e CD31 e CD63 CD81 e Y Rab5b e HLA G SO PEN 2OCK 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual Exosomes Culture Cells in Exosome depleted FBS FBS Removed Study exosomes from cultured cells and not from OW bovine exosomes in FBS itself Exo FBS has been stripped of bovine exosomes yet supports robust growth of cells in culture Exo Fgs l Exo FBS Pg Verify Exosome Recovery with Antibodies KDa mw F S Fd and Antibody Arrays 100 Track exosomes by Western blots and Antibody 70 Arrays using well characterized exosome protein 55 markers Verify exosome recoveries after isolation with ExoQuick or ultracentrifugation 35 using validated antibodies and arrays 25 Quantitate Exosomes with ELISAs ExoELISA CD9 Serum Plate poseen Exo ELISAs measure the levels of exosome qu particles with antibodies to detect CD9 CD63 or CD81 Highly sensitive and quantitative assays in a convenient 96 well format with validated sensitive and Accurate Quantitation exosome standards Se ow Amplify ExoRNA for Arrays M Un Amp 224 Page 16 ver 1 061114 www systembio com Amplify Exosome MicroRNAs with SeraMir for gPCR and microarrays Purify exoRNAs with SeraMir column
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