Human Reactive Exosome Marker Antibody Sampler Kit
Catalog# HAK21094
Human Reactive Exosome Marker Antibody Sampler Kit
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WB
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Human
General Overview
Kit Components
| Product Includes | Specification | Application | Reactivity | Mw |
|---|---|---|---|---|
| CD9[ET1601-9] | 20µl | WB,IF-Cell,IHC-P,IP,FC | Human,Mouse,Rat | Predicted band size: 25 kDa |
| TAPA1/CD81[ET1611-87] | 20µl | WB,IF-Cell,IHC-P,FC | Human,Mouse,Rat | Predicted band size: 26 kDa |
| CD63[HA722731] | 20µl | WB,IF-Cell,FC,IP,IHC-P | Human | Predicted band size: 26 kDa |
| TSG101[ET1701-59] | 20µl | WB,IF-Cell,IF-Tissue,IHC-P,FC | Human,Mouse,Rat | Predicted band size: 44 kDa |
| ALIX[ET1705-74] | 20µl | WB,IHC-P,FC,IF-Cell | Human,Mouse,Rat | Predicted band size: 96 kDa |
| Flotillin 1[ET7107-82] | 20µl | WB,IHC-P | Human,Mouse,Rat | Predicted band size: 47 kDa |
| Syntenin[ET7109-14] | 20µl | WB,IF-Cell,IF-Tissue,IHC-P,FC,IP | Human | Predicted band size: 32 kDa |
| Hsp70[ET1601-11] | 20µl | WB,IF-Cell,IF-Tissue,IHC-P,FC | Human,Mouse,Rat | Predicted band size: 70 kDa |
| Goat Anti-Rabbit IgG (H+L)[HA1001] | 100µl | WB,ELISA,IHC-P | Rabbit |
Product Description
The Human Reactive Exosome Marker Antibody Sampler Kit provides an economical means of analyzing proteins that can be present on exosomes.
Product Features
Storage Buffer
PBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Storage Instructions
Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Background
Exosomes are small (30-150 nm) membrane-bound vesicles that are secreted by various cell types under normal and pathological conditions. They originate from intracellular multivesicular endosomes upon fusion with the plasma membrane. Exosomes have emerged as an important mechanism of intercellular communication facilitating the transfer of membrane and cytosolic proteins, lipids, and RNA. Heterogeneity in exosome composition can be attributed to the cells of origin as well as the isolation methods. Tetraspanins are a family of cell surface glycoproteins with four transmembrane domains often found in exosomes. Tetraspanins CD9, CD81, and CD63 appear in exosomes and have been the target of immune-affinity approaches of exosome isolation. Flotillin-1 is a lipid raft-associated integral membrane protein that is incorporated into exosomes. Alix regulates cellular processes, such as endocytic membrane trafficking and cell adhesion through interactions with ESCRT proteins including endophilins, and CIN85 (Cbl-interacting protein of 85 kDa), and plays a role in exosome biogenesis . Syntenin-1 (MDA9, SDCBP) is a member of the PDZ family of proteins that functions as a scaffold adaptor protein regulating numerous signal transduction pathways. Syntenin-1 interacts with Alix to regulate exosome biogenesis. Tumor susceptibility gene 101 (TSG101) is a fundamental component of the ESCRT complex I involved in regulating the trafficking of proteins throughout the endosomal compartment. TSG101 is involved in regulating diverse biological processes, such as cell proliferation, viral budding and release, and exosome biosynthesis. The heat shock protein HSP70 is a molecular chaperone involved in protein folding that can be induced upon environmental stress. HSP70 may also be secreted through exosomes.
Data Links
Background References
1. Raposo, G. and Stoorvogel, W. (2013) J Cell Biol 200, 373-83.
2. van Niel, G. et al. (2018) Nat Rev Mol Cell Biol 19, 213-228.
3. Jeppesen, D.K. et al. (2019) Cell 177, 428-445.e18.
4. Kowal, J. et al. (2016) Proc Natl Acad Sci U S A 113, E968-77.
5. Sidhom, K. et al. (2020) Int J Mol Sci 21, 6466. doi: 10.3390/ijms21186466.
6. Patel, G.K. et al. (2019) Sci Rep 9, 5335.
7. Tauro, B.J. et al. (2012) Methods 56, 293-304.
8. Hemler, M.E. (2005) Nat Rev Mol Cell Biol 6, 801-11.
9. de Gassart, A. et al. (2003) Blood 102, 4336-44.
10. Katoh, K. et al. (2003) J Biol Chem 278, 39104-13.
Images
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Western blot analysis of CD9 on different lysates with Rabbit anti-CD9 antibody (ET1601-9) at 1/2,000 dilution.
Lane 1: HeLa cell lysate, 20 µg/Lane
Lane 2: K-562 cell lysate, 20 µg/Lane
Lane 3: MCF7 cell lysate, 20 µg/Lane
Lane 4: HCT 116 cell lysate, 20 µg/Lane
Lane 5: HepG2 cell lysate, 20 µg/Lane
Lane 6: SK-MEL-28 cell lysate, 20 µg/Lane
Lane 7: A375 cell lysate, 20 µg/Lane
Lane 8: B16-F1 cell lysate, 20 µg/Lane
Predicted band size: 25 kDa
Observed band size: 23 kDa
Exposure time: 3 minutes 30 seconds;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-9) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-CD9 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-9, 1/200) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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Western blot analysis of TAPA1/CD81 on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1611-87, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: PC-12 cell lysate
Lane 2: JAR cell lysate -
Western blot analysis of CD63 on different lysates with Rabbit anti-CD63 antibody (HA722731) at 1/1,000 dilution.
Lane 1: SK-MEL-28 cell lysate, 20 µg/Lane
Lane 2: Jurkat cell lysate (negative), 20 µg/Lane
Lane 3: U-87 MG cell lysate, 20 µg/Lane
Lane 4: HUVEC cell lysate, 20 µg/Lane
Lane 5: K-562 cell lysate, 20 µg/Lane
Lane 6: HEK-293 cell lysate, 20 µg/Lane
Lane 7: HL-60 cell lysate, 20 µg/Lane
Predicted band size: 26 kDa
Observed band size: 30-65 kDa
Exposure time: Lane 1: 4 seconds; Lane 2-7: 18 seconds; ECL: K1802;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722731) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
Immunohistochemical analysis of paraffin-embedded human melanoma tissue with Rabbit anti-CD63 antibody (HA722731) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA722731) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Flow cytometric analysis of K-562 cells labeling CD63.
Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (HA722731, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black). -
Western blot analysis of TSG101 on different lysates with Rabbit anti-TSG101 antibody (ET1701-59) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution.
Lane 1: HeLa cell lysate, 20 µg/Lane
Lane 2: K-562 cell lysate, 20 µg/Lane
Lane 3: MCF7 cell lysate, 20 µg/Lane
Lane 4: Jurkat cell lysate, 20 µg/Lane
Lane 5: C2C12 cell lysate, 20 µg/Lane
Lane 6: PC-12 cell lysate, 20 µg/Lane
Predicted band size: 44 kDa
Observed band size: 44/47 kDa
Exposure time: 3 minutes 20 seconds;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1701-59) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
Western blot analysis of TSG101 on different lysates with Rabbit anti-TSG101 antibody (ET1701-59) at 1/2,000 dilution.
Lane 1: HeLa cell lysate, 20 µg/Lane
Lane 2: K-562 cell lysate, 20 µg/Lane
Lane 3: Jurkat cell lysate, 20 µg/Lane
Lane 4: NIH/3T3 cell lysate, 20 µg/Lane
Lane 5: PC-12 cell lysate, 20 µg/Lane
Lane 6: Mouse brain tissue lysate, 20 µg/Lane
Lane 7: Rat brain tissue lysate, 20 µg/Lane
Predicted band size: 44 kDa
Observed band size: 44/47 kDa
Exposure time: 1 minute 40 seconds;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1701-59) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature. -
Immunocytochemistry analysis of PC-12 cells labeling TSG101 with Rabbit anti-TSG101 antibody (ET1701-59) at 1/200 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-TSG101 antibody (ET1701-59) at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-TSG101 antibody (ET1701-59) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1701-59) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Flow cytometric analysis of HeLa cells labeling TSG101.
Cells were fixed and permeabilized. Then stained with the primary antibody (ET1701-59, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black). -
Western blot analysis of ALIX on different lysates with Rabbit anti-ALIX antibody (ET1705-74) at 1/2,000 dilution.
Lane 1: HEK-293 cell lysate, 15 µg/Lane
Lane 2: K-562 cell lysate, 15 µg/Lane
Lane 3: Jurkat cell lysate, 15 µg/Lane
Lane 4: PC-3M cell lysate, 15 µg/Lane
Lane 5: MCF7 cell lysate, 15 µg/Lane
Predicted band size: 96 kDa
Observed band size: 100 kDa
Exposure time: 5 minutes;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1705-74) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature. -
Western blot analysis of ALIX on different lysates with Rabbit anti-ALIX antibody (ET1705-74) at 1/1,000 dilution.
Lane 1: NIH/3T3 cell lysate (20 µg/Lane)
Lane 2: PC-12 cell lysate (20 µg/Lane)
Lane 3: HEK-293 cell lysate (20 µg/Lane)
Lane 4: K-562 cell lysate (20 µg/Lane)
Lane 5: Mouse liver tissue lysate (30 µg/Lane)
Lane 6: Rat liver tissue lysate (30 µg/Lane)
Predicted band size: 96 kDa
Observed band size: 80-100 kDa
Exposure time: 6 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1705-74) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
Immunocytochemistry analysis of MCF7 cells labeling ALIX with Rabbit anti-ALIX antibody (ET1705-74) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-ALIX antibody (ET1705-74) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
Flow cytometric analysis of PC-12 cells labeling ALIX.
Cells were fixed and permeabilized. Then stained with the primary antibody (ET1705-74, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black). -
Western blot analysis of Flotillin 1 on different lysates with Rabbit anti-Flotillin 1 antibody (ET7107-82) at 1/1,000 dilution.
Lane 1: Neuro-2a cell lysate (20 µg/Lane)
Lane 2: Mouse brain tissue lysate (40 µg/Lane)
Lane 3: Mouse kidney tissue lysate (40 µg/Lane)
Lane 4: Mouse lung tissue lysate (40 µg/Lane)
Predicted band size: 47 kDa
Observed band size: 47 kDa
Exposure time: 59 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET7107-82) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-Hsp70 antibody (ET1601-11) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-11) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Western blot analysis of Hsp70 on different lysates with Rabbit anti-Hsp70 antibody (ET1601-11) at 1/2,000 dilution.
Lane 1: HeLa cell lysate, 15 µg/Lane
Lane 2: A549 cell lysate, 15 µg/Lane
Lane 3: MCF7 cell lysate, 15 µg/Lane
Lane 4: HCT 116 cell lysate, 15 µg/Lane
Lane 5: Mouse brain tissue lysate, 15 µg/Lane
Lane 6: Mouse testis tissue lysate, 15 µg/Lane
Lane 7: Rat testis tissue lysate, 15 µg/Lane
Predicted band size: 70 kDa
Observed band size: 70 kDa
Exposure time: Lane 1-4: 43 seconds; Lane 5-7: 2 minutes;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-11) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.
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