The suspension mentioned above was diluted with 5X sodium dodecyl sulfonate (SDS) buffer and was boiled for 10mins, in preparation for the following western blot analysis (10% SDS-polyacrylamide gel electrophoresis; 50 µg protein/lane) of two positive markers (CD63 and TSG101) and one negative marker (calnexin) of extracellular vesicles. Rabbit polyclonal antibody CD63 (sc-5275, Santa Cruz, CA, USA), TSG101 (sc-13611, Santa Cruz, CA, USA) and calnexin (10427-2-AP, Promega, Madison, WI) were used. The protein bands were detected using an enhanced chemiluminescence system (Bio-Rad, USA).
Tsg101 sc 13611
Manufactured by Santa Cruz Biotechnology
Sourced in United States
TSG101 (sc-13611) is a protein that plays a role in the endosomal sorting complex required for transport (ESCRT) pathway. It is involved in the formation of multivesicular bodies and the sorting of ubiquitinated proteins for lysosomal degradation.
Automatically generated - may contain errors
Lab products found in correlation
Cd63 sc 5275, by Santa Cruz Biotechnology (3 mentions)
Zetaview pmx 110, by Particle Metrix (3 mentions)
Calnexin 10427 2 ap, by Promega (2 mentions)
Alix sc 53540, by Santa Cruz Biotechnology (2 mentions)
H 7650, by Hitachi (2 mentions)
Enhanced chemiluminescence system, by Bio-Rad (1 mentions)
Amicon ultra spin filter, by Merck Group (1 mentions)
Hsp90 60318 1 ig, by Proteintech (1 mentions)
Cd9 60232 1 ig, by Proteintech (1 mentions)
Ab2787, by Abcam (1 mentions)
Ab275377, by Abcam (1 mentions)
Ab92573, by Abcam (1 mentions)
4 protocols using tsg101 sc 13611
1
Extracellular Vesicle Protein Analysis
2
Exosome Isolation and Characterization
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Exosomes were isolated by size exclusion chromatography. Briefly, 1 mL of 0.8 μm-filtered serum was diluted 1.5-fold with phosphate-buffered saline and further purified using Exosupur columns (Echobiotech, China) following the manufacturer’s instructions. Fractions were concentrated to 200 μL by 100 kDa molecular weight cutoff Amicon Ultra spin filters (Merck, Germany). The obtained exosome samples were observed by transmission electron microscopy, the particle size distribution was measured by nanoparticle tracking analysis using ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany), and their concentration was determined. The exosomes were also identified by western blot analysis using rabbit polyclonal antibody CD63 (sc-5275, Santa Cruz, CA, USA), CD9 (60232-I-Ig, Proteintech, Rosemont, IL), HSP90 (60318-I-Ig, Proteintech, Rosemont, IL), Alix (sc-53540, Santa Cruz, CA, USA), TSG101 (sc-13611, Santa Cruz, California) and calnexin (10427-2-AP, Promega, Madison, Wisconsin). The proteins were visualized on the Tanon4600 Automatic chemiluminescence image analysis system (Tanon, Shanghai, China).
3
Multimodal Exosome Characterization
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Exosomes were identified using three methods: nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blotting (WB). Initially, vesicle suspensions at concentrations of about 1 × 108/mL were analyzed using a ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) equipped with a 405 nm laser. The size and quantity of the isolated particles were determined. A total of 20 μL of exosomes was analyzed, and particle movement was assessed using NTA software (ZetaView 8.02.28). Subsequently, the exosomes were resuspended in PBS and placed on an electron microscope's copper mesh. After a 10-min incubation at room temperature, 1% uranyl acetate was used for negative staining for 10 min. Observations and images were made with a TEM (H-7650, Hitachi Ltd., Tokyo, Japan). Finally, exosome-enriched supernatant was denatured in 5 × SDS buffer and subjected to WB using TSG101 (sc-13611, Santa Cruz), HSP70 (ab2787, Abcam), CD63 (ab275377, Abcam), and Calnexin (ab92573, Abcam) [19 (link), 20 (link)].
4
Characterization of Small Extracellular Vesicles
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A total of 10 µL sEV‐enriched fraction was dropped on a copper mesh and incubated at 25°C for 10 min. The sEV‐enriched fraction was negative stained using uranyl acetate for 1 min. Subsequently, the sample was air dried for 2 min under incandescent light. Microphotographs were acquired using a transmission electron microscope (H‐7650, Hitachi Ltd., Tokyo, Japan).
Nanoparticle tracking analysis (NTA) was performed on the sEV‐enriched fractions using ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) equipped with a 405 nm laser module. The obtained data were analyzed using the corresponding software (ZetaView 8.02.28). To measure the size, concentration, and number of particles in the light‐scattering instrument, samples were diluted by PBS at concentrations between 1 × 107/mL and 1 × 109/mL.
Western blot analysis was performed to identify sEV‐positive (TSG101, sc‐13611, Santa Cruz, CA, USA; CD63, sc‐5275, Santa Cruz; and Alix, sc‐53540, Santa Cruz) and negative (calnexin, 10427‐2‐AP, Promega, Madison, WI, USA) markers. In brief, the sEV supernatant was denatured in 5× sodium dodecyl sulfonate (SDS) buffer and subjected to western blot analysis (10% SDS‐polyacrylamide gel electrophoresis; 50 µg protein/lane) using the above rabbit polyclonal antibodies. Proteins were then visualized on a Tanon 4600 automatic chemiluminescence image analysis system (Tanon, Shanghai, China).
Nanoparticle tracking analysis (NTA) was performed on the sEV‐enriched fractions using ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) equipped with a 405 nm laser module. The obtained data were analyzed using the corresponding software (ZetaView 8.02.28). To measure the size, concentration, and number of particles in the light‐scattering instrument, samples were diluted by PBS at concentrations between 1 × 107/mL and 1 × 109/mL.
Western blot analysis was performed to identify sEV‐positive (TSG101, sc‐13611, Santa Cruz, CA, USA; CD63, sc‐5275, Santa Cruz; and Alix, sc‐53540, Santa Cruz) and negative (calnexin, 10427‐2‐AP, Promega, Madison, WI, USA) markers. In brief, the sEV supernatant was denatured in 5× sodium dodecyl sulfonate (SDS) buffer and subjected to western blot analysis (10% SDS‐polyacrylamide gel electrophoresis; 50 µg protein/lane) using the above rabbit polyclonal antibodies. Proteins were then visualized on a Tanon 4600 automatic chemiluminescence image analysis system (Tanon, Shanghai, China).
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