antibodies and reagents for ev research · antibodies and reagents for ev research 55 exo-green kit...

Antibodies and reagents for EV research

Summary Section 6Introduction 54Exo-GREEN 55Antibodies for exosome research 57Antibody specifications 58

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Antibodies for Exosome researchAntibodies are an essen-tial tool for basic research, development of diagnostic tests and therapeutics for human disease. Extracellu-lar vesicles (EVs), including exosomes, express antigens with 3D conformations and/or post-translational modifi -cations that oft en diff er from the cellular counterpart. For this reason, most of the anti-bodies currently available on the market fail to recognize exosome-associated antigens with suffi cient sensitivity and

specifi city. HBM has validated a list of polyclonal and monoclonal antibodies against common (CD63, CD81, ALIX) and disease- specifi c (cancer and neuro-degenerative diseases) exosomal markers.

Exo-GREEN: Green fl uorescent dye for EV tracking

Together with the antibody panel, HBM-LS has devel-oped Exo-GREEN, a new re-agent for fl uorescent labelling isolated exosomes and mi-crovesicles that can be used for tracking EVs in fl uores-cence microscopy studies.

Products can be purchased directly in our

on-line shop:

www.exotest.eu/online_orders/

Distributors:

www.hansabiomed.eu/index.php/distribu-

tors

For information and techincal support

contact us at:

[email protected]

IntroductionOrdering information

Technical support

Catalog 2018Section 6Antibodies and reagents for EV research

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Exo-GREEN kit consists of a buff -ered lipid dye and a mini size ex-clusion chromatography (SEC) col-umn (see miniPURE-EVs, section 2). Th e fl uorescent probe, added to purifi ed vesicles, penetrates into the vesicle membrane, allowing a permanent and stable fl uorescent labeling. Vesicles are subsequently separated from the probe excess through a SEC mini column. Fluo-rescent-labeled vesicles are stable for long-term storage at -20°C (ap-proximately 6 months).

Cat. Code Reactions

Exo-GREEN: green fl uorescent dye for EV tracking

HBM-EGREEN-5 5 reactions

HBM-EGREEN-10 10 reactions

• Fluorescent labeling of pre-isolated

exosomes and microvesicles.

• EV-traking in cell biology

experiments.

• Long-term stability of labeled EVs

stored at -20°C.

• Adapt for labeling exosomes and

microvesicles isolated from diff erent

matrices.

• Kit is shipped at 4°C. Labeling probe

has to be stored at -20°C and can be

frozen and thawed multiple times.

Exo-GREEN: Green fl uorescent dye for EV tracking Applications

Advantages

Shipment and storage


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50 μg of purifi ed exosomes from BLCL21 cell media were treated with Exo-GREEN, following the manufac-turer’s instructions. Labeled Exosomes were subsequently analyzed by fl uo-rescence microscopy performed with Zeiss LSM Dou 510 META (Fig 1). FACS analysis was additionally performed, comparing Exo-GREEN-labeled exo-somes vs unlabeled and with purifi ed exosomes stained using CD9 antibody (Alexa 488) (Fig2). Aliquotes of labeled exosomes were stored at -80° and the fl uorescence was tested aft er 6 months.

Fresh purifi ed Exosomes Exo-GREEN labeled

Exosomes Exo-GREEN labeled stored 6 months at -80°C

1. Fluorescent microscopy of Exo-Green labeled exosomes

2. FACS analysis of purifi ed BLCL exosomes, labeled with CD9-Alexa488, with Exo-GREEN and unlabeled

Exo-GREEN stabiy labels Exosomes and Microvesicles for fl uorescence microscopy studies


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Cat. Code Antibody Type Applicartions

Antibodies for common Exosome Markers

HBM-TSG101-### Anti human TSG101 Mouse monoclonal unconjugated WB, IHC

HBM-CD9-### Anti human CD9 Mouse monoclonal unconjugated

WB, FACS, ELISA, IP, IHC

HBM-CD9B-### Anti human CD9 Mouse monoclonal biotin conjugated WB,, ELISA, IP

HBM-CD63-### Anti human CD63 Mouse monoclonal unconjugated


HBM-CD41-EM1-### Anti human CD41 Mouse monoclonal unconjugated FACS, ELISA

HBM-CD81-EM4-### Anti human CD81 Mouse monoclonal unconjugated


HBM-ALIX-### Anti human Alix Mouse monoclonal unconjugated WB, FACS, IF

HBM-FLOT-### Anti human Flotillin Rabbit polyclonal unconjugated

WB,FACS, ELISA

HBM-RAB5-PR1-### Anti human RAB5 Rabbit polyclonal unconjugated

WB, FACS, ELISA, IP

HBM-CD9M-### Anti mouse CD9 Mouse monoclonal unconjugated WB, FACS, ELISA, IP

Antibodies for Exosome-associated disease markers

HBM-HSP70-SR1-### Anti human HSP70 Rabbit polyclonal unconjugated

WB, FACS, ELISA

HBM-SF4-PR2-### Anti human TM9SF4 Rabbit polyclonal unconjugated

WB, IP

HBM-CD44-EM1-### Anti human CD44 Mouse monoclonal unconjugated WB, ELISA, IP

HBM-CAV1-D4-### Anti human Caveolin 1 Mouse monoclonal unconjugated WB, FACS

List of abbreviations:

ELISA: Enzyme-Linked Immunosorbent AssayFACS: Fluorescent-Activated Cell SortingIHC: ImmunohistochemistryIP: ImmunoprecipitationIF: ImmunofluorescenceWB: Western Blotting

• Exosome marker detection using

different techniques.

• Exosome immunocapture.

• All HBM-LS antibodies are tested

and validated for exosome antigen

reactivity.

• Available in 2 sizes: 50 μg and 100 μg.

• All HBM antibodies are shipped at

4°C. Storage conditions are described

in Antibody datasheet.

Antibodies for common and disease-associated EV markers Applications

Advantages

Shipment and storage


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Anti human TSG101 antibodyDescription TSG101, a 46 kDa protein, is the product of a recently identifi ed Tumor Susceptibility Gene whose

inactivation in mouse fi broblasts results in cell transformation and the ability of those cells to form tumors in nude mice. TSG101 is highly expressed internally to EVs and it is considered one of the common markers for exosome detection.

Cat Num/Amount HBM-TSG101-050 (50 μg)HBM-TSG101-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, IHC

Anti human CD9 antibodyDescription Anti-CD9 recognizes a human 24-kiloDalton (kDa) single-chain cell-surface glycoprotein (p24)

belonging to the tetraspanin family. CD9 has a very broad tissue distribution and is abun-dant on exosome membranes. HBM off ers monoclonal anti-CD9 antibodies, unconjugated or biotin conjugated, recognizing the specifi c antigen on both cell lines and extracellular vesicles.

Cat Num/Amount HBM-CD9-050 (50 μg)HBM-CD9-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, ELISA, FACS, IP, IHCReferences - Rampini, S., et al. “Micromagnet arrays for on-chip focusing, switching, and separation of superparamagnetic beads and

single cells.” Lab on a Chip 15.16 (2015): 3370-3379.- Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray plat-form.” Analytica Chimica Acta (2015).

Western Blott ing1- MM1 (melanoma cell) lysate (20 µg)2- MM1 cells purifi ed exosomes (20 µg)3-Plasma healthy donors purifi ed exosomes (20 µg)

ELISACD9 detection in purifi ed exosomes from human plasma (PEP) and urine (PEU), 30 µg

Western Blott ingDetection of TSG101 in 20 μg of Exosome Standards from:1-HBM-PES: healthy human serum2-HBM-HCT116: HCT116 cell culture medium3-RAMOS Exo: exosomes purifi ed from RAMOS cell medium4- HEK293 WT Exo: exosomes purifi ed from HEK293 wild type cell medium.

FACS CD9 staining of exosomes purifi ed from human plasma


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Anti human CD9 antibody biotin conjugatedDescription Anti-CD9 recognizes a human 24-kiloDalton (kDa) single-chain cell-surface glycoprotein (p24)

belonging to the tetraspanin family. CD9 has a very broad tissue distribution and is abundant on exosome membranes. HBM off ers two monoclonal anti-CD9 antibodies, unconjugated or biotin conjugated, recognizing the specifi c antigen on both cell lines and extracellular vesicles. HBM antibodies are compatible with ELISA, WB and fl ow cytometry applications.

Cat Num/Amount HBM-CD9B-050 (50 μg)HBM-CD9B-100 (100 μg)

Type Mouse monoclonal biotin conjugatedReactivity HumanApplications WB, ELISA, IPReferences - Rampini, S., et al. “Micromagnet arrays for on-chip focusing, switching, and separation of superparamagnetic beads and

single cells.” Lab on a Chip 15.16 (2015): 3370-3379.- Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray plat-form.” Analytica Chimica Acta (2015).

Anti human CD63 antibodyDescription Anti-CD63 recognizes an extracellular fragment of CD63, a 56 kiloDalton (kDa), type III lysosomal

glycoprotein, belonging to the tetraspanin family. CD63 is expressed by granulocytes, platelets, T-cells, monocytes/macrophages and endothelial cells. CD63 protein is a canonical exosome marker and currently used to characterize exosome populations from a variety of body fl uids.

Cat Num/Amount HBM-CD63-050 (50 μg)HBM-CD63-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, ELISA, FACS, IP, IHC

Western blott ing1- COLO1 cell lysates (20 µg)2- CD9 detection in plasma purifi ed exosomes (20 µg)

ELISACD9 detection in purifi ed exosomes from hu-man plasma (PEP) and urine (PEU), 30 µg

ELISA CD63 detection in purifi ed exosomes derived from cell supernatants (COLO1-30, MM1-30, BLCL21-30)



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Anti human CD41 antibody Description Integrin alpha chain 2b, also known as CD41, is an heterodimeric integral membrane protein. It

undergoes post-translational modifi cations that result in two polypeptide chains linked by a di-sulfi de bond. CD41 is expressed on platelets and megakaryocytes, but also on early embryonic hematopoietic stem cells and related exosomes.

Cat Num/Amount HBM-CD41-EM1-050 (50 μg)HBM-CD41-EM1-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications FACS, ELISA

Anti human Alix antibody Description Alix protein, named also ALG2 interacting protein or PDCD6-interacting protein, is a cytoplas-

mic protein that interacts with apoptosis associated-proteins. Alix plays an active role in exosome biogenesis and it is a useful internal marker for the analysis of exosomal proteins with western blott ing.

Cat Num/Amount HBM-ALIX-SM1-050 (50 μg)HBM-ALIX-SM1-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, FACS, IF

ELISAPEP-30: 30 ug of purifi ed exosomes from human healthy donors plasma.MM1-30: 30 ug of purifi ed exosomes from MM1 cell supernatantCOLO1-30: : 30 ug of purifi ed exosomes from COLO1 cell supernatant

Immunofl uorescenceImmunofl uorescence staining of HeLA cells.

Western blott ing1- MM1 (melanoma cell) lysate (20 ug)2- MM1 cells purifi ed exosomes (20 ug)3-Plasma healthy donors purifi ed exosomes (20 ug)



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Anti human CD81 antibodyDescription CD81 (TAPA1), a member of the tetraspanin family, is virtually expressed on all nucleated cells, but

in particular on germinal center B cells. CD81 forms complexes with other tetraspanin proteins, integrins and coreceptors. CD81 is expressed on exosome membranes. HBM anti-CD81 mono-clonal antibody is adapt for specifi c antigen recognition from cell lysates and exosomes using the techniques indicated below.


Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, ELISA, FACS, IP, IHCReferences - Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray plat-

form.” Analytica Chimica Acta (2015).

Anti human Flotillin antibodyDescription Flotillin belongs to the band 7.2/stomatin protein family and appears to be strongly expressed in

muscle cells and fi broblasts. Flotillin expression is also correlated with Alzheimer development. Flo-tillin is highly expressed on exosomes and appears to be involved in exosome release mechanism. It is considered a common marker for exosomes analyses.

Cat Num/Amount HBM-FLOT-SR1-050 (50 μg)HBM-FLOT-SR1-100 (100 μg)

Type Rabbit polyclonal unconjugatedReactivity HumanApplications WB, ELISA, FACSReferences Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray plat-


Western blott ing1- MM1 (melanoma cell) lysate (20 ug)2- MM1 cells purifi ed exosomes (20 ug)3-Plasma healthy donors purifi ed exo-somes (20 ug)

FACS CD81 staining of COLO1 cell purifi ed exosomes

ELISACD81 on purifi ed exosomes from urine (PEU), 30 ug

FACS Purifi ed exosomes from MM1 (30 ug)

stained by anti-Flotillin antibody

ELISA Detection of diff erent exosomes purifi ed from cell culture supernatants or human plasma (HD), performed with anti-Flotillin


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Anti human Rab5 antibodyDescription Rab5 is a small GTPase belonging to Ras superfamily of monomeric G proteins. Rab GTPases play

an essential role in the regulation of membrane traffi c and are involved in vesicle formation and transport and fusion to the membrane. Rab5 is expressed on exosome membranes and it might have an active role during endo/exocytotic processes of microvesiscles through the plasma mem-brane.

Cat Num/Amount HBM-RAB5-PR1-050 (50 μg)HBM-RAB5-PR1-100 (100 μg)

Type Rabbit polyclonal unconjugatedReactivity HumanApplications WB, ELISA, FACS, IPReferences Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray plat-


Anti human TSTA3 antibodyDescription Tissue specifi c transplantation antigen P35B is a NADP(H)-binding protein. It catalyzes the two-

step epimerase and the reductase reactions in GDP-D-mannose metabolism, converting GDP-4-keto-6-D-deoxymannose to GDP-L-fucose. TSTA3 has been identifi ed in the overall exosome population.

Cat Num/Amount HBM-TSTA3-PR1-050 (50 μg)HBM-TSTA3-PR1-100 (100 μg)

Type Rabbit polyclonal unconjugatedReactivity HumanApplications WB, ELISA

Western blott ingExosomes purifi ed from 0.5, 0.25, 0.1 ml of hu-man plasma demonstrate diff erent intensity of Rab5 expression

0.5 ml 0,25 ml 0,1 ml

FACS Purifi ed exosomes from COLO1 cell line detected by Rab5 anti-body

Western blott ingDetection of TSTA3 in:20 ug COLO1 purifi ed exosmes. 20 ug of exosomes purifi ed human plasma (PEP)*in purifi ed exosomes from plasma TSTA3 can ap-pear in dimer form (signal around 70 kDa)


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Anti mouse CD9 antibodyDescription Anti-CD9 recognizes a human 24-kiloDalton (kDa) single-chain cell-surface glycoprotein

(p24) belonging to the tetraspanin family. CD9 has a very broad tissue distribution and is abundant on exosome membranes. HBM off ers a monoclonal anti-CD9 antibody unconju-gated reactive against mouse antigen and able to identify CD9 in mouse derived exosomes.

Cat Num/Amount HBM-CD9M-050 (50 μg)HBM-CD9M-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity MouseApplications WB, ELISA, FACS, IP, IHC

FACS CD9 staining of B16F10 mouse cell pu-rifi ed exosomes

1 2

Western blott ing1- 20ug of whole lysate of B16F10 cell super-natant purifi ed exosomes 2- 20 ug of exosomes isolated from mouse plasma


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Anti human Caveolin antibodyDescription Th e lipid raft -associated protein caveolin-1 (CAV1) is the major component of the inner surface

of caveolae, small invaginations of the plasma membrane. Caveolin is a transmembrane adaptor molecule that can simultaneously recognize GPI-linked proteins and interact with downstream cytoplasmic signaling molecules. It is highly expressed on exosomes derived from tumor tissue.

Cat Num/Amount HBM-CAV1-D4-050 (50 μg)HBM-CAV1-D4-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, FACS

Anti human CD44 antibody Description CD44, known also as HCAM, is a 742 aminoacid protein involved in lymphocyte activation, hom-

ing and hematopoiesis, CD44 is expressed in multiple isoforms. CD44 is highly expressed in cancer tissues and tumor-derived exosomes, suggesting a role in tumor progression and metastasis.


Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, ELISA, IP

FACSStaining of Cav1 in MM1 purifi ed exosomes

Western blott ing 1-20 ug of whole lysate of melanoma cell derived exosomes (MM1) 2- 20 ug of whole MM1 cell lysate

Western blott ing1- 20ug of whole lysate of MM1 cell super-natant purifi ed exosomes 2- 20 ug of purifi ed exosomes from human plasma of healthy donors (PEP)

ELISAPEP-30: 30 ug of purifi ed exosomes from human healthy donors plasma.MM1-30: 30 ug of purifi ed exosomes from MM1 cell supernatantCOLO1-30: : 30 ug of purifi ed exosomes from COLO1 cell supernatant


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Anti human TM9SF4 antibodyDescription TM9SF4 (TUCAP1) is a newly discovered tumor-associated protein of unknown function that

belongs to the Trans-Membrane 9 Superfamily (TM9SF). Th ese proteins are characterized by the presence of a large variable extracellular N-terminal domain followed by nine putative transmem-brane domains and a conserved C-terminal domain. TM9SF4 is mainly expressed on exosomes derived from tumor tissue. HBM off ers three diff erent monoclonal antibodies that recognize the protein on both exosomes and cell lysates.

Cat Num/Amount HBM-SF4-PMG1-050 (50 μg)HBM-SF4-PMG1-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, FACS, IP

Anti human HSP70 antibodyDescription Heat shock protein 70 (HSP70) is a molecular chaperone that facilitates the assembly of multi-

protein complexes and traffi cking of polypeptides across cell membranes. HSP70 is active in pro-moting tumorigenesis and functions as an anti-apoptotic factor. It is highly expressed on exosomes derived from tumor tissues.

Cat Num/Amount HBM-HSP70-SR1-050 (50 μg)HBM-HSP70-SR1-100 (100 μg)

Type Rabbit polyclonal unconjugatedReactivity HumanApplications WB, ELISA, FACS, IPReferences Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray plat-


HBM-MM1 (Melanoma cell

Exosome Standards)

HBM-PEP(Plasma healthy donor Exosome Standards)

Western blott ing1- 20 ug of whole lysate of melanoma cell line2- 20ug of whole lysate of MM1 cell superna-tant purifi ed exosomes 3- 20 ug of purifi ed exosomes from human plasma of healthy donors (PEP)

FACSStaining of TM9SF4 on MM1 purifi ed exosomes vs human plasma exosomes (PEP) demonstrates the predominant TM9SF4 expression in exosomes derived from tumoral source

Western blott ingAnalysis of HSP70 expression in diff erent exosomes derived from tumoral cell lines:1- COLO1; 2- SH-SYSY; 3 - U87; 4 - SK

FACS Staining of COLO1 derived exosomes with anti-HSP70


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antibodies and reagents for ev research · antibodies and reagents for ev research 55 exo-green kit...

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