Tsg101 c 2

Manufactured by Santa Cruz Biotechnology

TSG101 (C-2) is a monoclonal antibody that recognizes the TSG101 (Tumor Susceptibility Gene 101) protein. TSG101 is a cellular protein involved in various cellular processes, including cell cycle regulation, endosomal trafficking, and viral budding.

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Lab products found in correlation

Cfp c192, by American Type Culture Collection (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Animage quant las4000, by GE Healthcare (1 mentions) Cd81 h 121, by Santa Cruz Biotechnology (1 mentions) Rabbit anti mouse igg, by GeneTex (1 mentions) Goat anti rabbit igg, by GeneTex (1 mentions) Hsc70 b6, by Santa Cruz Biotechnology (1 mentions) Calnexin, by Cell Signaling Technology (1 mentions) Mhc 1, by Abcam (1 mentions) Cd63 h 193, by Santa Cruz Biotechnology (1 mentions) α tubulin antibody, by Abcam (1 mentions) Lamp1 1d4b, by Abcam (1 mentions) His tag, by Santa Cruz Biotechnology (1 mentions) Sc 17768, by Santa Cruz Biotechnology (1 mentions) Ubiquitin p4d1, by Santa Cruz Biotechnology (1 mentions)

5 protocols using tsg101 c 2

Isolation and Analysis of Extracellular Vesicles

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To prepare brain homogenates, tissue was removed from the frontal lobe,
lysed in strong lysis buffer (detailed above) with proteinase inhibitor,
homogenized by pipette, and centrifuged at 10,000 × g for 10 minutes to
discard whole cells and intact cellular debris. For confirmation of EV proteins
in purified lysates, 5X Laemmli sample buffer [10% SDS, 250 mM Tris pH 6.8, 1
mg/mL bromophenol blue, 0.5 M DTT, 50% glycerol, 5% BME] was added to EV
isolates for a final concentration of 1X. For immunoblot analysis of CD63, DTT
and BME were omitted from lysis and sample buffers to allow the protein to run
in non-reducing conditions. Gel electrophoresis and western blotting was
performed as previously described (Hurwitz et
al., 2017b). Equal volume of gradient lysates were loaded into an
SDS-PAGE gel. Blots were probed with the following antibodies: Alix (Q-19; Santa
Cruz Biotechnology), HSC70 (B-6; Santa Cruz), TSG101 (C-2; Santa Cruz), CD63
(TS63; Abcam), Rab8a (63-BJ ; Santa Cruz), and CD81 (H-121 ;Santa Cruz), rabbit
anti-mouse IgG (Genetex, 26728), rabbit anti-goat IgG (Genetex, 26741), goat
anti-rabbit IgG (Fab fragment) (Genetex, 27171). Blots were imaged using an
Image Quant LAS4000 (General Electric) and processed with ImageQuant TL v8.1.0.0
software, Adobe Photoshop CS6 and CorelDraw Graphic Suite X5.

Hurwitz S.N., Sun L., Cole K.Y., Ford CR I.I.I., Olcese J.M, & Meckes DG J.r. (2018). An optimized method for purification of whole brain-derived extracellular vesicles reveals insight into neurodegenerative processes in a mouse model of Alzheimer’s disease.. Journal of neuroscience methods, 307, 210-220.

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Western Blotting of Cell Lysates

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Western blotting was performed as previously described[6 (link)] on samples lysed in RIPA buffer, using the following antibodies: flotillin-1 (C-2, SantaCruz, 0.4μg/ml), MHC-I (HLA-A; EP1395Y, Abcam, 0.103μg/ml), TSG101 (C-2, SantaCruz, 1μg/ml), calnexin (#2433, CellSignallingTechnologies, 0.8μg/ml).

Rice T.F., Donaldson B., Bouqueau M., Kampmann B, & Holder B. (2018). Macrophage- but not monocyte-derived extracellular vesicles induce placental pro-inflammatory responses. Placenta, 69, 92-95.

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Extracellular Vesicle Protein Analysis

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EV pellets were resuspended in 50 µl of lysis buffer (0.15 M NaCl, 5 mM EDTA, pH 8, 1% Triton X-100, and 10 mM Tris-HCl, pH 7.4), and protein contents were quantified using a BCA protein assay kit according to the manufacturer’s protocol (Thermo Fisher Scientific). Protein extracts from EV pellets and cell lysates were prepared with Laemmli buffer in nonreductive conditions, heated at 95°C for 5 min, and separated on 15% polyacrylamide gels before transfer to a nitrocellulose membrane (Hybond ECL plus; Thermo Fisher Scientific). Membranes were blocked in 5% nonfat milk and incubated with the anti-CD9 (H-110; Santa Cruz Biotechnology, Inc.), -CD63 (H-193; Santa Cruz Biotechnology, Inc.), -TSG101 (C-2; Santa Cruz Biotechnology, Inc.), LAMP1 (1D4B; Abcam), and α-tubulin antibodies (4074; Abcam) followed by the horseradish peroxidase–coupled secondary antibody and then were subjected to enhanced chemiluminescence.

Stik G., Crequit S., Petit L., Durant J., Charbord P., Jaffredo T, & Durand C. (2017). Extracellular vesicles of stromal origin target and support hematopoietic stem and progenitor cells. The Journal of Cell Biology, 216(7), 2217-2230.

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Exosome Protein Profiling by Immunoblotting

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Exosomes (10 μg or 10⁹ particles) were resuspended in PBS with protease inhibitors. The suspension was mixed with Laemmli buffer, heated at 95 °C for 5 min, and chilled on ice for 5 min before loading onto SDS gel. Immunoblots probed with antibodies for proteins: Tsg-101 (C-2, 1:1000, Santa Cruz), CFP (C192, 1:1000, ATCC), 19kDa-lipoprotein (IT-19, 1:1000, ATCC), His-tag (1:500, Santa Cruz), CD81 (1:500, SBI), CD63 (1:500, Systems Bioscience), and, DsRed (632393, 1:1000, Clontech). Primary antibody incubation was followed with HRP-conjugated secondary antibodies (1:25,000, Pierce) and detected using enhanced chemiluminecence kit (Pierce). As loading controls, primary mouse mAbs against either alpha-Tubulin, 1/1000 dilution (Cat. T9026, Sigma) or Lamp-1 1/500 dilution (SC-17768, Santa Cruz Biotechnology or 1D4B, DHSB) were used.

Smith V.L., Cheng Y., Bryant B.R, & Schorey J.S. (2017). Exosomes function in antigen presentation during an in vivo Mycobacterium tuberculosis infection. Scientific Reports, 7, 43578.

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Exosome Protein Profiling by Western Blot

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Exosomes (10 μg) were resuspended in 1X PBS with protease inhibitors. The suspension was mixed with Laemmli buffer, heated at 95°C for 5 min, and chilled on ice for 5 min before loading onto SDS gel. Whole cell lysates were prepared in RIPA buffer. Suspension was mixed with Laemmli buffer, heated at 95°C for 5 min. before loading onto SDS gel. Immunoblots probed with antibodies for proteins: ubiquitin (P4D1, 1:1000, Santa Cruz), Tsg101 (C-2, 1:1000, Santa Cruz), Hrs (V-20, 1:500, Santa Cruz), Tubulin (T5293, Sigma), CFP (C192, 1:1000, ATCC), Kat-G (IT-42, 1:20, ATCC), His (1:500, Santa Cruz), GroES (SA-12, 1:20, ATCC), HspX (IT-20, 1:15, ATCC), Park2 (ARP43038, 1:500, Aviva). Primary antibody incubation was followed with HRP-conjugated secondary antibodies (1:25,000, Pierce) and detected using enhanced chemiluminecence kit (Pierce).

Smith V.L., Jackson L, & Schorey J.S. (2015). Ubiquitination as a mechanism to transport soluble mycobacterial and eukaryotic proteins to exosomes. Journal of immunology (Baltimore, Md. : 1950), 195(6), 2722-2730.

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