Anti-TSG101 antibody

• WB

Project

Western blot - Anti-TSG101 antibody (AB30871)

All lanes:

Western blot - Anti-TSG101 antibody (ab30871) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

A431 (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 3:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 4:

HEK293 Human embryonic kidney cell line Whole Cell Lysate at 10 µg

Lane 5:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 6:

MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Lane 7:

SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate at 10 µg

Lane 8:

U2OS (Human osteosarcoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 44 kDa

Observed band size: 49 kDa

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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-TSG101 antibody (AB30871)

ab30871 stained in HeLa cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab30871 at 5μg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 μM for 1hour at room temperature.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TSG101 antibody (AB30871)

IHC image of ab30871 staining in human placenta formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab30871, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• WB

Lab

Western blot - Anti-TSG101 antibody (AB30871)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab30871 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

The doublet seen in Western blot has been described in PMID : 11427703 and may be due to internal initiation at Met₁₀.

All lanes:

Western blot - Anti-TSG101 antibody (ab30871) at 1 µg/mL

Lane 1:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Lane 2:

PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 44 kDa

Observed band size: 29 kDa,48 kDa,49 kDa

true

Exposure time: 90s

• WB

CiteAb

Western blot - Anti-TSG101 antibody (AB30871)

Western Blotting using Anti-TSG101 antibody, ab30871. Publication image from van Rheenen, J. et al., 2020, Nat Commun, 32111843. Legend direct from paper.

Intercellular sgRNA exchange is EV-mediated.a Schematic of EV isolation workflow and analysis. Conditioned medium is isolated from sgRNA+ MDA-MB-231 donor cells, after which cell debris is pelleted through centrifugation. Conditioned medium is then concentrated by tangential flow filtration, followed by size exclusion chromatography-mediated EV isolation. EVs are then characterized, and used for EV addition experiments in Stoplight+spCas9+ reporter cells. b Nanosight Nanoparticle Tracking Analysis (NTA) showing the size distribution of the isolated EVs (mode = 81 ± 3 nm). c Western blot analysis of EVs and cell lysates for common EV markers (CD9, CD63, ALIX, Flot-1, and TSG101), and EV-negative markers Calnexin and H2B. Representative images as observed in three independent experiments. d Electron microscopy analysis of isolated EVs. Scale bar represents 100 nm. Representative image as observed in 12 separate fields. e An EV RNase protection assay, followed by RT-PCR sgRNAs, shows that sgRNAs are only susceptible to RNase-mediated degradation in the presence of the membrane-disrupting anionic surfactant SDS. Representative data as observed in three independent experiments. f Confocal microscopy images of HEK293T reporter cells (red) that have taken up MDA-MB-231-derived PKH67-labeled EVs (green). Representative image as observed in seven randomly selected fields. g, h EV-mediated activation of the CROSS-FIRE platform using EVs isolated from sgRNA+ MDA-MB-231 donor cells. EVs were added every 72 h for 12 additions with an average dose of 1.1e11 ± 4.9e10 EVs per addition. Cells were analyzed by fluorescence microscopy (g) and flow cytometry (h). Scale bar represents 200 µm. Means + SD, n = 3 biological replicates, Tukey’s multiple comparison test. i EVs from sgRNA+ MDA-MB-231 donor cells activate Stoplight+ spCas9+ HEK293T reporter cells in a dose-dependent manner. EVs were added every 72 h for nine additions with an average concentration of 2.2e10 ± 1e10 (20 µl) or 2.2e11 ± 1e11 (200 µl). Means + SD, n = 3 biological replicates, Tukey’s multiple comparison test. *p < 0.05.

false

• WB

CiteAb

Western blot - Anti-TSG101 antibody (AB30871)

Western Blotting using Anti-TSG101 antibody, ab30871. Publication image from Yang, G. Y. et al., 2021, Theranostics, 33391532. Legend direct from paper.

Characterization of M2 BV2 cells induced by IL-4 and identification of sEVs derived from M2 BV2 cells. (A, B) Representative images of BV2 cells immunostained for Iba-1 (green), CD206 (red), and arginase (red). Cultured systems were treated with 0 or 20 ng/µL IL-4. Cell nuclei were counterstained with DAPI. Scale bar = 50 µm. (C) Western blotting analysis of CD206 and arginase expression in BV2 cells after 0 or 20 ng/µL IL-4 treatment. (D) Representative electron microscopy images showing the phenotype of M2-sEVs. Left image scale bar = 100 nm, right image scale bar = 50 nm. (F) NTA of M2-sEVs isolated by ultracentrifugation from M2 BV2 cells. Data represent the average size distribution profile of three samples and each purification normalized to the total nanoparticle concentrations. Data for each sample was derived from three different videos and analyses. (G) Western blotting analysis of TSG101 and CD63 levels in M2 BV2 cells and M2-sEVs.

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