Anti-Mesothelin antibody [EPR28269-62]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Mesothelin antibody [EPR28269-62] (AB309516)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Tween-20 permeabilized NIH OVCAR-3 (human ovary adenocarcinoma epithelial cell) cells labelling Mesothelin with ab309516 at 1/100 (5.08 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing mainly membranous staining in NIH : OVCAR-3 cell line.Negative control : A431 (PMID : 33262421).Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt

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Flow Cytometry - Anti-Mesothelin antibody [EPR28269-62] (AB309516)

Flow cytometric analysis of A431 (human epidermoid carcinoma epithelial cell, Left) / NIH : OVCAR-3 (human ovary adenocarcinoma epithelial cell, Right) cells labelling Mesothelin with ab309516 at 1/50 dilution (1 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody. Negative control : A431(PMID : 33262421). Gated on viable cells.

• IP

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Immunoprecipitation - Anti-Mesothelin antibody [EPR28269-62] (AB309516)

Mesothelin was immunoprecipitated from 0.35 mg NIH OVCAR-3 (human ovary adenocarcinoma epithelial cell) whole cell lysate with ab309516 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab309516 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : NIH OVCAR-3 (human ovary adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : ab309516 IP in NIH OVCAR-3 (human ovary adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab309516 in NIH : OVCAR-3 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 5 seconds

All lanes:

Immunoprecipitation - Anti-Mesothelin antibody [EPR28269-62] (ab309516) at 1/1000 dilution

All lanes:

NIH OVCAR-3 (human ovary adenocarcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 5s

• WB

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Western blot - Anti-Mesothelin antibody [EPR28269-62] (AB309516)

Blocking and diluting buffer and concentration : 5% NFDM/TBST 69 kDa full length band and 40 kDa cleavage fragment band were ovserved. Exposure time : Lane 1 : 169 seconds Lane 2 : 70 seconds

All lanes:

Western blot - Anti-Mesothelin antibody [EPR28269-62] (ab309516) at 1/1000 dilution

Lane 1:

Human ovary cancer tissue lysate at 20 µg

Lane 2:

HeLa (human epithelial cell line from cervical adenocarcinoma) whole cell lysate at 20 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 1/100000 dilution

Observed band size: 49 kDa,69 kDa

false

Exposure time: 169s

• WB

Supplier Data

Western blot - Anti-Mesothelin antibody [EPR28269-62] (AB309516)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : A431(PMID : 20922056); MCF-7(PMID : 8552591). 69 kDa full length band and 40 kDa cleavage fragment band were ovserved. The expression molecular weight observed is consistent with what has been described in the literature (PMID : 20922056; PMID : 8552591) Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : Lane 1, 2 : 48 seconds Lane 3, 4 : 70 seconds

All lanes:

Western blot - Anti-Mesothelin antibody [EPR28269-62] (ab309516) at 1/1000 dilution

Lane 1:

NIH OVCAR-3 (human ovary adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH:OVCAR-3 whole cell lysate at 20 µg

Lane 4:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 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 1/100000 dilution

Observed band size: 40 kDa,69 kDa

false

• WB

Supplier Data

Western blot - Anti-Mesothelin antibody [EPR28269-62] (AB309516)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : Huh-7 (PMID : 20922056). 69 kDa full length band and 40 kDa cleavage fragment band were ovserved. This blot was developed using a higher sensitivity ECL substrate. Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 3 minutes

All lanes:

Western blot - Anti-Mesothelin antibody [EPR28269-62] (ab309516) at 1/1000 dilution

Lane 1:

SK-OV-3 (human ovarian cancer epithelial cell) whole cell lysate at 20 µg

Lane 2:

Huh7 (human hepatocellar carcinoma epithelial cell) whole cell lysate 20 at 20 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 1/100000 dilution

Observed band size: 40 kDa,69 kDa

false

Exposure time: 3min