Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Bicinchoninic acid (BCA) protein assay kit and Imperial Protein stain was from Pierce (Thermo Scientific, Rockford, IL, USA). Mini-protean TGX 4-20% polyacrylamide gels and Transfer-Blot Turbo Transfer Pack were from Bio-Rad (Hercules, CA, USA). PVDF membranes were from Millipore (Billerica, MA, USA). The antibodies used for Western blotting were: mouse anti-Tsg101 (BD Biosciences, Heidelberg, Germany); rabbit anti-CD9 (Abcam, Cambridge, UK); mouse anti-CD81 (Ancell Corporation, Bayport, MN, USA), rabbit anti-uromodulin (St. Cruz Biotechnology Inc., Dallas, TX, USA). HRP-conjugated secondary antibodies were from Jackson Immunoresearch (West Grove, PA, USA). The antibodies used for immuno-electron microscopy were: mouse anti-CD63 (H5C6) (DSHB, Iowa city, IA, USA) and rabbit-anti-mouse (DACO, Glostrup, Denmark). Protein A-gold conjugates (10 nm) were purchased from Cell Microscopy Center (Utrecht, Netherlands).
Mouse anti tsg101
Manufactured by BD
Sourced in United States
Mouse anti-TSG101 is a primary antibody that recognizes the tumor susceptibility gene 101 (TSG101) protein. TSG101 is a ubiquitin-conjugating enzyme involved in the trafficking of proteins from the cell surface to the lysosome for degradation. This antibody can be used to detect and quantify TSG101 expression in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti alix, by Cell Signaling Technology (3 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
Optiprep, by Axis-Shield (2 mentions)
Goat anti rabbit igg h l hrp conjugate, by Bio-Rad (2 mentions)
Mini protean tgx 4 20 polyacrylamide gels, by Bio-Rad (1 mentions)
Transfer blot turbo transfer pack, by Bio-Rad (1 mentions)
Rabbit anti cd9, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rabbit anti gapdh, by Abcam (1 mentions)
Mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Bolt 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ibind flex system, by Thermo Fisher Scientific (1 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Vectastain abc hrp kit, by Vector Laboratories (1 mentions)
Anti mouse igg, by Vector Laboratories (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Irdye 800cw donkey anti mouse igg, by LI COR (1 mentions)
Intercept blocking buffer, by LI COR (1 mentions)
10 protocols using mouse anti tsg101
1
Extracellular Vesicle Protein Characterization
2
Western Blot Analysis of Cell/EV Samples
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Western blot analyses of cell/EV samples (10 μg protein) were performed as described [26 (link)]. Briefly, membranes were probed with primary mouse anti-TSG101 (1:500, BD Biosciences, San Jose, CA), mouse anti-Alix (1:1000, Cell Signalling Technology Danvers, MA), mouse anti-CD9 antibody (1:1000, Abcam, Cambridge, MA), mouse anti-KIF23 (1:1000, Thermo Fisher Scientific, CA), and rabbit anti-GAPDH (1:1000, Abcam, Cambridge, UK) in TTBS (Tris buffered-saline containing 0.1% Tween-20) for 1 h. After washing with TTBS (3 × 10 min), membranes were probed with secondary antibody (IRDye 800 goat anti-mouse or IRDye 700 goat anti-rabbit IgG, 1: 15,000). The fluorescent signals were detected using the Odyssey Infrared Imaging System, v3.0 (Li-COR Biosciences, Nebraska, USA).
3
EV Protein Extraction and Western Blot
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Protein extraction was performed using mPER (Pierce) with resultant protein concentration evaluated by BCA (Pierce) following the manufacturer’s instructions. EV aliquots containing approximately 1 × 1011 particles were incubated in an equal volume of mPER. For each EV sample, 20 million “parent” cells (that had generated the EV) were resuspended in 1 mL of mPER. Protein samples were frozen at −20 °C until used. 4 μg of EV-derived or matched cell-derived protein was separated using Bolt 4–12% Bis-Tris gels and MES SDS running buffer (ThermoFisher). Protein was transferred onto a nitrocellulose membrane using an iBlot 2 dry blotting system (ThermoFisher) set to 15 V for 15 minutes. Ab labeling was performed using the iBind Flex system (ThermoFisher) using primary mouse anti-CD9 [MM2/57] (BioRad MCA469GA) or mouse anti-TSG101 (BD 612696) and anti-mouse IgG (Vector) secondary Ab. Positive bands were detected using the Vectastain ABC HRP kit and DAB peroxidase substrate kit (Vector) per the manufacturer’s instructions.
4
EV Protein Characterization by SDS-PAGE and Immunoblotting
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For SDS‐PAGE, a volume of EVs corresponding to 1.5 μg of total protein was prepared in Laemmli sample buffer (BioRad 1610747) containing β‐mercaptoethanol and heated at 95°C for 5 minutes. Proteins were separated through a 4%–12% NuPAGE Bis‐Tris gel (Invitrogen), transferred to an Immobilon‐FL PVDF membrane (Millipore), and detected as described previously (Yang et al., 2011 (link)). After blocking with Intercept blocking buffer (Licor Biosciences), the following antibodies were used: mouse anti‐calnexin (BD Biosciences 610523, 1:500), rabbit anti‐GM130 (Abcam ab52649, 1:500), mouse anti‐cytochrome c (BD Biosciences 556433, 1:1000), rabbit anti‐CD9 (Cell Signaling Technologies 13174, 1:1000), mouse anti‐TSG101 (BD Biosciences 612696, 1:500), and mouse anti‐THP (Santa Cruz Biotechnology sc‐271022, 1:1000), rabbit anti‐beta‐actin (Invitrogen, PA1‐16889, 1:5000), donkey anti‐mouse IgG IRDye 800CW (LiCor 925–32212, 1:10,000), and donkey anti‐rabbit IgG IRDye 680RD (LiCor 926–68073, 1:10,000).
5
Protein Quantification by SDS-PAGE/Densitometry
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Sample protein concentrations were determined using one-dimensional SDS-PAGE/SYPRO Ruby protein staining/densitometry, as described [5] (link), [6] (link). Briefly, samples were lysed in SDS sample buffer for 20 min at room temperature, and proteins (10 µg/sample) resolved by SDS-PAGE, electrotransferred onto nitrocellulose membranes using the iBlot Dry Blotting System (Life Technologies), and membranes blocked with 5% (w/v) skim milk powder in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TTBS) for 30 min. Membranes were probed overnight in TTBS with primary mouse anti-CD9 (at 1∶1000) and mouse anti-TSG101 (at 1∶1,000) from BD Biosciences, mouse anti-Alix (at 1∶1,000) from Cell Signalling, or mouse anti-A33 (1 µg/ml) (gift from Dr A Scott, Ludwig Institute for Cancer Research, Melbourne). After washing with TTBS (3×10 min) membranes were incubated with horse radish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma) or IRDye 800 anti-mouse IgG (Li-COR Biosciences). Proteins were visualized by incubating membranes with Western HRP substrate (Merck-Millipore) followed by imaging with ChemiDocMP System (Bio-Rad) or by imaging directly with the Odyssey Infrared Imaging System (v3).
6
Immunostaining of Retinal Pigment Epithelial Cells
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Calcium and magnesium free PBS (PBS−) was purchased from Gibco (#10010-023). Triton X-100 was obtained from Sigma-Aldrich (#T8787). Hoechst 33258 (#H3569) and AlexaFluor 568-conjugated Phalloidin (#A12380) were from Invitrogen. Antibodies used were as follows: Mouse anti-RPE65 (#ab78036 [clone 401.8B11.3D9]; Abcam, Cambridge, MA), mouse anti-Cytokeratin (#M0821 [clone MNF116]; Dako A/S, Glostrup, Denmark), rabbit anti-ZO1 (mid) (#40-2200; ThermoFisher Scientific, Waltham, MA), mouse anti-Syntenin-1 (#ab131190 [clone 3D9-G9-H4]; Abcam), mouse anti-TSG101 (#612696; BD Biosciences, San Jose, CA), rabbit anti-SLC39A12 (#ab106570; Abcam), rabbit anti-Calreticulin (#12238 [clone D3E6]; Cell Signaling Technologies, Danvers, MA), mouse anti-BEST1 (#NB300-164, [clone E6-6]; Novus Biologicals, Littleton, CO), mouse anti-Na+/K+-ATPase alpha (#sc-58628 [clone M7-PB-E9]; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-CD36 (#ab78054; Abcam), AlexaFluor 488-conjugated donkey-anti-rabbit IgG (#A21206, Invitrogen), AlexaFluor 488-conjugated donkey-anti-mouse IgG (#A21202, Invitrogen), AlexaFluor 568-conjugated donkey-anti-rabbit IgG (#A10042; Invitrogen), HRP-conjugated donkey-anti-mouse IgG (#715-035-150, Jackson ImmunoResearch Laboratories, West Grove, PA), and HRP-conjugated donkey-anti-rabbit IgG (#711-035-152, Jackson ImmunoResearch Laboratories).
7
Characterization of Extracellular Vesicles by WB
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The following anti-human antibodies and their dilution rates were used for western blot (WB) analyses: mouse anti-CD63 (Abcam, Cambridge, MA, USA; 1:1,000), mouse anti-CD133 (Abnova Corp., Taipei city, Taiwan; 1:1,000), rabbit anti-aquaporin 5 (AQP5) (Abcam; 1:500), mouse anti-β-actin (Sigma–Aldrich, St. Louis, MO, USA; 1:1,000), rabbit anti-CD26 (Abcam; 1:1,000), mouse anti-CD81 (EXBIO, Plaha, Czech; 1:1,000), mouse anti-EpCAM/TROP-1 (R&D Systems, Minneapolis, MN, USA; 1:1,000), rabbit anti-HSP70 (System Biosciences, Mountain View, CA, USA; 1:1,000), mouse anti-TSG101 (BD Transduction Laboratories, San Jose, CA, USA; 1:500), rabbit anti-CD44 (Sigma–Aldrich; 1:1,000), rabbit anti-CD24 (Santa Cruz Biotechnology, Dallas, TX, USA; 1:100) and mouse anti-CD9 (Abcam; 1:500). Secondary antibodies coupled to horseradish peroxidase were as follows: goat anti-rabbit IgG (H+L)-HRP conjugate (Bio-Rad, Hercules, CA, USA; 1:2,000), goat anti-mouse IgG [(H+L)-HRP conjugate (Bio-Rad; 1:2,000)] and rabbit anti-goat IgG (H+L)-HRP conjugate (Bio-Rad; 1:2,000). Iohexol (Nycodenz) and iodixanol (OptiPrep) purchased from Axis-Shield PoC (Oslo, Norway) were used as density gradient media. Each gradient medium was prepared in 0.02 M HEPES [4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid]/NaOH, pH 7.2 buffer.
8
Antibody Production and Characterization
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Antiserum against a peptide corresponding to amino acids 25–39 of human CHMP6 was raised in two rabbits and affinity purified on Affi-Gel beads containing immobilized peptide. Rabbit anti-CHMP3, rabbit anti-ALIX, rabbit anti-EAP30, rabbit anti-CHMP4B, and rabbit anti-EAP20 were described previously (Sharma et al., 2004 (link); Cabezas et al., 2005 (link); Bache et al., 2006 (link); Malerød et al., 2007 (link); Sagona et al., 2010 (link)). Mouse anti–α-tubulin (Sigma-Aldrich), sheep anti–α-tubulin (Cytoskeleton), mouse anti–β-actin (Sigma-Aldrich), goat anti-RacGAP1 (Abcam), rabbit anti-MKLP1 (Santa Cruz Biotechnology, Inc.), goat anti-V5 (Abcam), mouse anti-GFP (Roche), mouse anti-TSG101 (BD Transduction Laboratories), rabbit anti-CHMP6 (Santa Cruz Biotechnology, Inc.), rabbit anti-CEP55 (Abnova), rabbit anti-VPS28 (Santa Cruz Biotechnology, Inc.), rabbit anti-IST1 (Proteintech), rabbit anti-CHMP4A (Santa Cruz Biotechnology, Inc.), and rabbit anti-mCherry (Acris) were used as primary antibodies. Secondary antibodies included anti-mouse, anti-rabbit, and anti-goat Alexa Fluor 488 (Jackson ImmunoResearch), Alexa Fluor 555 (Molecular Probes), Alexa Fluor 568 (Molecular Probes), Alexa Fluor 647 (Jackson ImmunoResearch), and DyLight649 (Jackson ImmunoResearch).
9
Quantitative Protein Profiling of EVs
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Protein content was estimated by 1D-SDS-PAGE/SYPRO® Ruby protein staining densitometry, as previously described173 (link). For immunoblotting (10 μg), membranes were probed with primary antibodies [mouse anti-TSG101 (BD Transduction Laboratories; 1:500), mouse anti-Alix (Cell Signaling Technology; 1:1000), rabbit anti-CD70 (Abcam; 1:1000), mouse anti-HSPG1 (Abcam; 1:200), rabbit anti-MSLN (Cell Signaling Technology; 1:1000), rabbit anti-FAT1 (GeneTex; 1:2000), mouse anti-CD81 (Santa Cruz Biotechnology; 1:1000), rabbit anti-CALR (Abcam; 1:1000) for 3 hr at room temperature (RT) in 50 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween 20 (TTBS) followed by incubation with either IRDye 800 goat anti-mouse or goat IgG or IRDye 680 goat anti-rabbit IgG (1:15000, LI-COR Biosciences) for 1 hr at RT in TTBS. Immunoblots were visualized using the Odyssey Infrared Imaging System and Image Studio™ Software (v3.0, LI-COR Biosciences, Nebraska USA).
10
Extracellular Vesicle Characterization in Colorectal Cancer
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SW480 cells were from Ludwig Institute for Cancer Research Ltd. (Parkville Branch, Melbourne) and SW620 cells were from Dr E. Vincan (Peter MacCallum Cancer Centre, Australia). All media and supplements were from Life Technologies (NY, USA). OptiPrep™ was from Axis-Shield PoC (Norway). CELLine AD-1000 Bioreactor classic flasks were from Integra Biosciences. Mouse anti-Alix, anti-CD44, rabbit anti-MET, anti-GAPDH were from Cell Signaling, Sigma-Aldrich (MA, USA), Mouse, anti-EGFR, anti-CD9, anti-CD63, rabbit anti-MET, anti-PAK1, anti-CLDN1, anti-ANXA1 and goat-anti-CLDN7 were from Santa Cruz Biotechnology (CA, USA) and Mouse anti-TSG101 was from BD Transduction Laboratories (NJ, USA).
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