Tsg101

Manufactured by System Biosciences

Sourced in United States

TSG101 is a protein involved in the endosomal sorting complex required for transport (ESCRT) machinery. It plays a critical role in the sorting and trafficking of ubiquitinated proteins into multivesicular bodies.

Automatically generated - may contain errors

Lab products found in correlation

Hsp70, by System Biosciences (12 mentions) Fei spirit 120kv, by Thermo Fisher Scientific (4 mentions) Fv1000, by Olympus (4 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions) Mini protean tgxtm gel, by Bio-Rad (2 mentions) Ecl system, by Merck Group (2 mentions) Postn, by Abcam (2 mentions) Gapdh, by Abcam (2 mentions) Vegfa, by Santa Cruz Biotechnology (2 mentions) Bca protein assay, by Beyotime (2 mentions) Chemiluminescent substrate system, by Thermo Fisher Scientific (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Nitrocellulose filter membrane, by Merck Group (2 mentions) Free protease inhibitor cocktail, by Roche (2 mentions) Hmox1, by Abcam (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Ripa buffer, by Beyotime (2 mentions) Chemidoc xrs system, by Bio-Rad (2 mentions) Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Beyotime (2 mentions)

10 protocols using tsg101

Western Blot Analysis of Exosomal Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols

For western analysis, samples were mixed with the 5 × SDS sample buffer and boiled at 95 °C for 10 min. Samples were then separated by 10% SDS-PAGE gel (Mini-PROTEAN TGXTM Gels, Bio-Rad) and transferred to nitrocellulose membrane. Membranes were blocked with 5% nonfat milk in TBST buffer (Intron), and probed with a primary antibody. After washing membranes with TBST four times, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody in TBST with 5% nonfat milk. After washing, protein bands were visualized using the ECL system (Millipore).
Following antibody dilutions were used for western analysis: Hsp70 (SBI, rabbit), 1:1000~1500; TSG101 (SBI, rabbit), 1:1000; CD63 (SBI, rabbit), 1:1000; CD81 (SBI, rabbit), 1:500~1,000; CD81 (Santa Cruz, mouse), 1:200; HRP (SBI, rabbit or mouse), 1:10,000.

Lee S.S., Won J.H., Lim G.J., Han J., Lee J.Y., Cho K.O, & Bae Y.K. (2019). A novel population of extracellular vesicles smaller than exosomes promotes cell proliferation. Cell Communication and Signaling : CCS, 17, 95.

+ Open protocol

+ Expand

Isolation and Characterization of Tumor-Derived Exosomes

Check if the same lab product or an alternative is used in the 5 most similar protocols

Tumor cells were cultured in RPMI 1640 media supplemented with 10% exosome-depleted FBS (BI, Israel). Supernatant of tumor cells culture was collected 48 hours after cell reached 80% confluence. Then the supernatant was centrifuged at 4000 rpm for 2 hours to remove cell debris, followed by 4000 rpm centrifuge for 30 min using 100 KDa MWCO to make the exosome-concentrated solution. The exosome was isolated by exosome quick extraction solution. The protein content of exosome was determined by BCA protein assay kit. The characterization of exosomes was confirmed by measuring expression of exosome-specific markers ALIX, HSP70, TSG101, CD81 and CD9 (SBI) by western blot analysis and Transmission Electron Microscopy (FEI spirit 120kV, USA). Exosomes were labeled with PKH67 membrane dye at 37°C for 5 minutes, and analyzed by confocal microscope (FV1000, Olympus). All the unspecified exosomes used in this study were from either TC-1 or MC38 tumor cells.

Yin X., Zeng W., Wu B., Wang L., Wang Z., Tian H., Wang L., Jiang Y., Clay R., Wei X., Qin Y., Zhang F., Zhang C., Jin L, & Liang W. (2020). PPARα Inhibition Overcomes Tumor-Derived Exosomal Lipid-Induced Dendritic Cell Dysfunction. Cell reports, 33(3), 108278.

+ Open protocol

+ Expand

Isolation and Characterization of Tumor-Derived Exosomes

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

Exosome Protein Characterization Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed in 1% n-octyl-p-D-glucopyranoside (OG) buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% OG, 1 mM EDTA, 10 g/ml leupeptin, 2 g/ml aprotinin, 1 mM PMSF). The total protein density was determined using bicinchoninic acid (BCA) protein assay kit (synthgene, China).The protein was then incubated overnight with the following primary antibodies at 4°C: CD9, CD63 and TSG101 (1:500, SBI, USA), POSTN (1:1500, Abcam, USA), GAPDH (1:2000, Abcam, USA). GAPDH served as a loading control.
After incubation with the corresponding second antibodies, protein bands were quanti ed using Image J Software.

Zhang J., Li Z., Liu C., Wu Y., Li C., & Meng J. (2021). LncRNA H19 from T-Regulatory-Cell-Derived Exosomes Sponges miR-154-5p to Promote Osteogenic Differentiation of BMMSCs Through Upregulating POSTN.

+ Open protocol

+ Expand

Exosome Characterization by Western Blot

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total protein was extracted using the Total Protein Extraction Kit (SAB, USA) according to the manufacturer’s instructions. Protein concentration was quantified using a BCA protein assay (Beyotime). Sixty micrograms of total protein were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to 0.22 µm polyvinylidene difluoride (PVDF) membranes at 200 mA for 1 h. The membranes were blocked with 10% albumin bovine (BSA, Biofroxx, Germany) for 1 h at room temperature. The blots were probed with primary antibodies for 24 h at 4 °C and further incubated with secondary antibodies for 1 h at room temperature. These primary antibodies included CD63 (System Biosciences, 1:1000), TSG101 (System Biosciences, 1:1000), ADM (Santa Cruz Biotechnology, USA, 1:1000), VEGFA (Santa Cruz Biotechnology, 1:1000), and NDRG1 (Santa Cruz Biotechnology, 1:1000). The supernatant of RP-Exos extract was used as a negative control for exosome characterization.

Li Y., Lyu P., Ze Y., Li P., Zeng X., Shi Y., Qiu B., Gong P, & Yao Y. (2021). Exosomes derived from plasma: promising immunomodulatory agents for promoting angiogenesis to treat radiation-induced vascular dysfunction. PeerJ, 9, e11147.

+ Open protocol

+ Expand

Exosome Protein Profiling by Western Blot

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell or exosome lysates obtained with RIPA lysis buffer (Thermo Fisher Scientific) were centrifuged at 13 200 rpm and 4°C for 15 minutes, and were quantified by the BCA method. Proteins were run on a SDS‐PAGE gel according to a standard protocol and were transferred onto a PVDF membrane (Merck Millipore). The PVDF membrane was blocked with 5% skim milk, and was sequentially incubated with the primary and the second antibodies. The following antibodies were used according to the manufacturer's instructions: CD63 (CST), CD9 (CST), TSG101 (System Biosciences), EGFR (CST), Cyclin E (CST), and β‐actin (Sigma‐Aldrich Co). Protein bands on PVDF membrane were detected using the Chemiluminescent Substrate System (Thermo Fisher Scientific). β‐actin was used as the internal control.

Shang S., Wang J., Chen S., Tian R., Zeng H., Wang L., Xia M., Zhu H, & Zuo C. (2019). Exosomal miRNA‐1231 derived from bone marrow mesenchymal stem cells inhibits the activity of pancreatic cancer. Cancer Medicine, 8(18), 7728-7740.

+ Open protocol

+ Expand

Characterizing EV Protein Concentration

Check if the same lab product or an alternative is used in the 5 most similar protocols

The protein concentration of EVs (n=4) was tested by Micro BCA protein analysis as described in the instructions (Thermo Scientific Pierce 23235). Expression of the EVs markers CD63 and TSG101 (System Biosciences) was verified by Western blot analysis (n=1).

Luo L., Yan C., Fuchi N., Kodama Y., Zhang X., Shinji G., Miura K., Sasaki H, & Li T.S. (2021). Mesenchymal stem cell-derived extracellular vesicles as probable triggers of radiation-induced heart disease. Stem Cell Research & Therapy, 12, 422.

+ Open protocol

+ Expand

Western Blot Analysis of Prostate Cancer Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed in RIPA buffer (Beyotime, China) containing protease inhibitors (Complete Mini Ethylene Diamine Tetraacetic Acid-Free Protease Inhibitor Cocktail, Roche). The protein lysates were centrifuged, and the supernatants were collected for protein quantification with a Bicinchoninic Acid assay kit (Pierce, USA). Protein lysates were resolved with 8% to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Beyotime, China), and were electrophoretically transferred to nitrocellulose filter membranes (Millipore). The membranes were then probed with antibodies against prostate-specific antigen (PSA) (1:1000; Cell Signaling Technology), AR (1:1000; Cell Signaling Technology), TSG101 (1:1000; System Biosciences), HSP70 (1:1500; System Biosciences), Alix (1:1000; System Biosciences) and HMOX1 (1:1000; Abcam). GAPDH (1:800; Cell Signaling Technology) and β-actin (1:1000; Cell Signaling Technology) were used as loading controls. After incubation with horseradish peroxidase (HRP)–conjugated goat anti-rabbit immunoglobulin G or HRP-conjugated goat anti-mouse immunoglobulin G secondary antibodies, the proteins were observed with the ChemiDoc XRS+ system (Bio-Rad, USA).

Zhang Y., Chen B., Xu N., Xu P., Lin W., Liu C, & Huang P. (2021). Exosomes Promote the Transition of Androgen-Dependent Prostate Cancer Cells into Androgen-Independent Manner Through Up-Regulating the Heme Oxygenase-1. International Journal of Nanomedicine, 16, 315-327.

+ Open protocol

+ Expand

Exosomal Protein Profiling Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Exosomes were lysed with RIPA buffer [50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 0.5% deoxycholate, 0.1% SDS, and 1.0% NP-40] containing protease inhibitor cocktail (Roche). Protein lysates (20 μg) were loaded onto a 4–20% Tris–glycine gradient gel (Biorad), transferred onto PVDF membranes and Western blotting was performed as per standard protocols. Following transfer, membranes were cut according to predicted molecular sizes and probed with following antibodies: THBS1 (Cell Signaling Technology, 37879), CD9 (Cell Signaling Technology, 13174), CD63 (System Biosciences, EXOAB-CD63A-1), Alix (Cell Signaling Technology, 2171), TSG101 (System Biosciences, EXOAB-TSG101-1) and Gelsolin (Cell Signaling Technology, 12953).

Bhagirath D., Liston M., Akoto T., Lui B., Bensing B.A., Sharma A, & Saini S. (2021). Novel, non-invasive markers for detecting therapy induced neuroendocrine differentiation in castration-resistant prostate cancer patients. Scientific Reports, 11, 8279.

+ Open protocol

+ Expand

Characterization of Exosome Ultrastructure

Check if the same lab product or an alternative is used in the 5 most similar protocols

Image of exosomes were taken by a transmission electron microscope. Brie y, 5 ul exosomes were dropped on the copper net and incubated at room temperature (RT) for 5 minutes. Then, excess liquids were removed by the lter paper. Add 5 ul 1% phosphotungstic acid to the copper mesh and incubated for 1 minute at RT. Excess liquids were also removed by the lter paper. Add deionized water to the copper mesh to remove excess dye solution. Observe the exosomes under microscope after drying at RT. The protein concentration of exosomes was tested as described in the instructions (Thermo Scienti c 23235). Expression of exosome marker CD63 and TSG101 (System Biosciences) were veri ed western blot analysis. Exosome miRNAs were analyzed using gene chip miRNA 4.0 by the Filgen company.

Luo L., Chen Y., Fuchi N., Kodama Y., Zhang X., Goto S., Miura K., Sasaki H., & Li T. (2021). Mesenchymal Stem Cells-derived Exosomes as Probable Triggers of Radiation-induced Heart Disease.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!