Anti tsg101 sc 7964

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-Tsg101 (sc-7964) is a primary antibody product offered by Santa Cruz Biotechnology. This antibody is designed to detect the Tsg101 (tumor susceptibility gene 101) protein. Tsg101 is a protein that plays a role in various cellular processes, including cell growth, division, and trafficking. The core function of this antibody is to specifically recognize and bind to the Tsg101 protein, allowing for its detection and analysis in various experimental applications.

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9 protocols using anti tsg101 sc 7964

Immunoblotting Analysis of Extracellular Vesicles

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Immunoblotting was performed using manufacturer protocols and standard conditions. The EV pellet was lysed using RIPA buffer with 1X Protease and Phosphatase Inhibitor Cocktail. Protein was assayed using the BCA protein assay. Protein lysates were mixed with 4X non-reducing lithium dodecyl sulfate sample loading buffer containing 3% 2-mercaptoethanol. Twenty micrograms total protein were loaded per lane of a gradient 4–12% precast SDS-PAGE gel (NuPAGE®, Life Technologies). Following separation, proteins were transferred to a nitrocellulose membrane then blocked with 5% bovine serum albumin (BSA) before being incubated with primary antibodies. The primary antibodies used included: anti-Flag (F1804, Sigma-Aldrich), anti-His (D3I1O, Cell Signaling), anti-TSG101 (SC7964, Santa Cruz) anti-calreticulin (CALR, 2891S, Cell Signaling) and anti-GAPDH (SC-32233, Santa Cruz Biotechnology, Dallas, TX). The membrane was washed using 0.05% Tween-20 in 1X TBS-T wash buffer then incubated with 1% BSA in TBS-T and secondary antibody (goat anti-mouse or goat-anti-rabbit) for 1 h. Presence of proteins was visualised using an enhanced chemiluminescence detection system (GE Healthcare Bio-Sciences, Pittsburgh, PA). GAPDH and TSG101 protein were used as loading controls for cells and EVs, respectively.

Sutaria D.S., Jiang J., Elgamal O.A., Pomeroy S.M., Badawi M., Zhu X., Pavlovicz R., Azevedo-Pouly A.C., Chalmers J., Li C., Phelps M.A, & Schmittgen T.D. (2017). Low active loading of cargo into engineered extracellular vesicles results in inefficient miRNA mimic delivery. Journal of Extracellular Vesicles, 6(1), 1333882.

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Detailed Antibody and Lipid Reagents

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Anti-VPS13D (A304-691A, Bethy Laboratories. Inc), Anti-GFP (AE011, Abclonal), anti-Halo (G9211; Promega), anti-Tubulin (100109-MM05T; Sinobiological), anti-actin (20536-1-AP; Proteintech), anti-VDAC1 (55259-1-AP; Proteintech), anti-TSG101 (A1692; Abclonal) were used at 1:1000 dilutions for western blots. Anti-VPS13D (A304-691A, Bethy Laboratories. Inc) and anti-TSG101 (SC7964; Santa Cruz Biotechnology) antibodies were used 1:100 for immunofluorescence. The following reagents were used in this study: Oleic acid (O1008; sigma); Palmitic acid (P0500; sigma); Doxycycline (1225984; Sigma), BODIPY 493/503 (ThermoFisher Scientific, D3922), BODIPY 558/568 (ThermoFisher Scientific, D2219), NBD-C12 (Abcam, Ab145361), BODIPY 558/568 C12 (ThermoFisher Scientific, D3835), BODIPY FL C16 (ThermoFisher Scientific, D3821), Janilia Fluo® 646 HaloTag® Ligand (GA1120; Promega) and antibiotics such as G418 (10131027) and puromycin (A1113803) were obtained from ThermoFisher Scientific. All EM reagents were purchased from Electron Microscopy Sciences. Lipids were purchased from Avanti Polar Lipids: NBD-PC (810133), NBD-PE (810144), NBD-PS (810198), NBD-PA (810138), NBD-ceramide (810211).

Wang J., Fang N., Xiong J., Du Y., Cao Y, & Ji W.K. (2021). An ESCRT-dependent step in fatty acid transfer from lipid droplets to mitochondria through VPS13D−TSG101 interactions. Nature Communications, 12, 1252.

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Characterization of Extracellular Vesicle Proteins

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EV proteins were extracted adding RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 2mMsodium fluoride, 2mMEDTA, and 0.1% SDS) containing a mixture of protease inhibitors (aprotinin, phenylmethylsulfonyl fluoride, and sodium orthovanadate) to the isolated EV pellets. Protein concentration was quantified by BCA Protein Assay Kit (Thermo Scientific, UK), according with manufacturer’s instructions. Equal amounts of EV extracts were resolved on 11% SDS–polyacrylamide gel as previously described [50 (link)], and probed with anti-Tsg101 (sc-7964, Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD9 (ab236630, Abcam, Cambridge, UK), anti-CD63 (ab213092, Abcam), anti-CD81 (ab155760, Abcam), anti-Alix (ab186429, Abcam) and anti-Calnexin (sc-11397, Santa Cruz Biotechnology) overnight at 4 °C, followed by secondary IRDye 800CW goat anti-mouse or anti-rabbit antibodies (LI-COR, Germany). The images were acquired using Odissey FC (LI-COR, Lincoln, NE, USA).

Barone I., Gelsomino L., Accattatis F.M., Giordano F., Gyorffy B., Panza S., Giuliano M., Veneziani B.M., Arpino G., De Angelis C., De Placido P., Bonofiglio D., Andò S., Giordano C, & Catalano S. (2023). Analysis of circulating extracellular vesicle derived microRNAs in breast cancer patients with obesity: a potential role for Let-7a. Journal of Translational Medicine, 21, 232.

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Western Blot Analysis of EV Markers

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MSC-exos, cells and kidney tissues were lysed in RIPA lysis buffer (ThermoFisher) and the protein concentration was detected using BCA assay (ThermoFisher). Protein samples were subjected to Bis-Tris Gel (Invitrogen) and transferred onto PVDF membranes (Millipore). Membranes were blocked in NcmBlot blocking buffer (NCM Biotech) for 10 min and incubated overnight with primary antibodies as follows: anti-CD9 (ab92726, Abcam), anti-CD63 (sc-5275, Santa Cruz), anti-Tsg101 (sc-7964, Santa Cruz) anti-Alix (sc-53540, Santa Cruz), anti-GM130 (12480, Cell Signaling Technology), anti-CD44 (ab189524, Abcam), anti-ICAM-1 (MA5407, Invitrogen), anti-VCAM-1 (39036, Cell Signaling Technology), anti-LFA-1 (ab13219, Abcam), anti-integrin α4 (8440, Cell Signaling Technology), anti-integrin β1 (sc-374429, Santa Cruz), anti-PCNA (sc-56, Santa Cruz), anti-CDK1 (sc-54, Santa Cruz), anti-caspase-3 (ab13847, Abcam), anti-Cyclin B1(4138, Cell Signaling Technology), anti-p53 (2524, Cell Signaling Technology), anti-Bcl-2 (sc-7382, Santa Cruz), and anti-Bax (sc-20067, Santa Cruz). Secondary antibodies were used for detection by an ECL advanced system (GE Healthcare). Intensity values expressed as the relative protein expression were normalized to β-actin (AB2001, Abways) or GAPDH (AB2000, Abways).

Cao J.Y., Wang B., Tang T.T., Wen Y., Li Z.L., Feng S.T., Wu M., Liu D., Yin D., Ma K.L., Tang R.N., Wu Q.L., Lan H.Y., Lv L.L, & Liu B.C. (2021). Exosomal miR-125b-5p deriving from mesenchymal stem cells promotes tubular repair by suppression of p53 in ischemic acute kidney injury. Theranostics, 11(11), 5248-5266.

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Western Blot Analysis of Cellular Proteins

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Western blotting was conducted using fresh cell lysates, which were separated via 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Sigma, St. Louis, MOUSA) that were then blocked for 1 to 2 h with 1% bovine serum albumin (BSA). The blots were probed overnight with appropriate primary antibodies at 4 ℃, followed by probing for 1 h with secondary horseradish peroxidase (HRP)-conjugated antibodies. Protein bands were then detected with an enhanced chemiluminescence (ECL) kit (Biosharp). The primary antibodies used were as follows: anti-calnexin (2679, Cell Signaling Technology, Danvers, MA, USA), anti-HSP70 (4872, Cell Signaling Technology), anti-TSG101 (sc-7964, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD9 (ab92726, Abcam, Cambridge, UK), anti-PTEN (9552, Cell Signaling Technology), anti-PI3K (4249, Cell Signaling Technology), anti-AKT (9272, Cell Signaling Technology), anti-P-AKT (4060, Cell Signaling Technology), and anti-GAPDH (10494-1-AP, Proteintech, Wuhan, China). Goat anti-rabbit IgG secondary antibody (HRP; Proteintech) and goat anti-mouse IgG (HRP; Proteintech) were used.

Xie X., Qu P., Wu H., Liu P., Luo J., Chi J., Liu X., Chen X, & Xu C. (2022). Circulating exosomal miR-21 mediates HUVEC proliferation and migration through PTEN/PI3K/AKT in Crohn’s disease. Annals of Translational Medicine, 10(5), 258.

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Western Blot Antibody Validation

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We used the following commercial antibodies for Western blot analyses: anticalnexin (Sc-6465), anti-CD81 (Sc-166029), and anti-Tsg101 (Sc-7964) from Santa Cruz Biotechnology; anti-CD9 (553758) and anti-Flotillin (610820) from BD Biosciences; anti-FLAG (F3165) from Sigma; anti-Histone H2A.Z (2718) from Cell Signaling; antilactate dehydrogenase (AB1222) from Millipore; and antivimentin (20R-VP004) from Fitzgerald. The antisyntenin antibody used in this study was affinity purified further from antisyntenin (Santa Cruz Biotechnology, sc-48742). We used anti-F-actin (abcam, ab205) for coimmunoprecipitation.

Qiu X., Campos Y., van de Vlekkert D., Gomero E., Tanwar A.C., Kalathur R., Weesner J.A., Bongiovanni A., Demmers J, & d’Azzo A. (2022). Distinct functions of dimeric and monomeric scaffold protein Alix in regulating F-actin assembly and loading of exosomal cargo. The Journal of Biological Chemistry, 298(10), 102425.

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Characterizing Extracellular Vesicle Proteins

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The protein concentration was determined with a BCA kit, and 30 μg of total protein was resolved by 10 % SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). Membranes were blocked with 5 % nonfat milk for 2 h and then incubated with primary antibodies (diluted at 1:1000) overnight at 4 °C. After washing three times with TBST, the membranes were added to the corresponding horseradish peroxidase–conjugated secondary antibodies (dilution ratio of 1:5000) and incubated for 1 h. After washing three times with TBST, a chemiluminescent reagent (Tanon, China) was dropped onto the membranes. Finally, the protein expression was observed with a chemiluminescence gel imaging analyzer (Tanon, China). The antibodies were anti-CD9 (AF5139, Affinity Biosciences, USA), anti-CD81 (DF2306, Affinity Biosciences, USA), anti-TSG101 (sc-7964, Santa Cruz Biotechnology, USA), anti-CD63 (sc-365604, Santa Cruz Biotechnology, USA), anti-CD73 (ab313339, Abcam, UK), and anti-β-actin (bs-0061R, Bioss Biotechnology, Bioss, China).

Zhang S., Li J., Li C., Xie X., He J., Ling F., Li B., Wu H., Li Z., Zhen J, & Liu G. (2023). CD73-positive pediatric urethral mesenchymal stem-like cell-derived small extracellular vesicles stimulate angiogenesis. Regenerative Therapy, 25, 77-84.

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Immunoblotting Analysis of Protein Complexes

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Cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 50 mM Tris [pH 8.0]) supplemented with 1μg/ml Aprotinin, 1mM DTT, 0.2mM PMSF 1μg/ml pepstatin A. The protein concentration was determined by Bradford Protein Assay (BioRad) and equal amount of proteins were resolved by SDS gel electrophoresis, transferred to a nitrocellulose membrane and subjected to a standard immunoblotting protocol. For the analysis of SEC eluted fractions, the proteins were isolated by Trichloroacetic Acid (Sigma-Aldrich) precipitation followed by acetone washes of the protein pellets. Precipitated proteins were re-suspended directly in Laemmli Sample buffer and resolved by SDS gel electrophoresis. The antibodies used were: rabbit polyclonal anti-Aurora A/AIK #3092, rabbit monoclonal anti-SMAD3 #9523, anti-SMAD2 #5339 (Cell Signaling); mouse monoclonal anti-Tsg101 sc-7964, anti-TGFβ RII sc-17791, anti-Fibronectin sc-8422, anti-Perforin 1 sc-373943, anti-HSP70 sc-24, anti-Alix sc-53540 and rabbit polyclonal anti-calnexin sc-11397 (Santa Cruz Biotechnology, Inc.); mouse monoclonal anti-MYCN Ab16898 and rabbit polyclonal anti-TGFβ RI Ab31013 (abcam); rabbit polyclonal anti-CD81 EXOAB-CD81A-1 (System Biosciences); mouse monoclonal anti-Granzyme A #507202 and anti-Granzyme B #674302 (Biolegend).

Neviani P., Wise P.M., Murtadha M., Liu C.W., Wu C.H., Jong A.Y., Seeger R.C, & Fabbri M. (2018). Natural Killer-derived exosomal miR-186 inhibits neuroblastoma growth and immune escape mechanisms.. Cancer research, 79(6), 1151-1164.

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Exosome Proteome Analysis Reagents

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Anti-Tsg101 (sc-7964) and anti-Calnexin (sc-11397) were acquired from Santa Cruz Biotechnology (Dallas, TX, USA); anti-ALIX (EPR14314) was acquired from Abcam (Cambridge, UK). Leptin and Exosome-depleted FBS were obtained from Life Technologies (Monza, MB, Italy). Phorbol 12-myristate 13-acetate (PMA) was purchased from Merck (Burlington, MA, USA).

Gelsomino L., Barone I., Caruso A., Giordano F., Brindisi M., Morello G., Accattatis F.M., Panza S., Cappello A.R., Bonofiglio D., Andò S., Catalano S, & Giordano C. (2022). Proteomic Profiling of Extracellular Vesicles Released by Leptin-Treated Breast Cancer Cells: A Potential Role in Cancer Metabolism. International Journal of Molecular Sciences, 23(21), 12941.

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