Anti-Prothrombin antibody [EPR20159]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Prothrombin antibody [EPR20159] (AB208589)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling Prothrombin with ab208589 at 1/600 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Prothrombin antibody [EPR20159] (AB208589)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling Prothrombin with ab208589 at 1/100 dilution followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HepG2 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• IP

Lab

Immunoprecipitation - Anti-Prothrombin antibody [EPR20159] (AB208589)

Prothrombin was immunoprecipitated from HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab208589 at 1/50 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab208589 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-Prothrombin antibody [EPR20159] (ab208589) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate (Input) at 10 µg

Lane 2:

HepG2 whole cell lysate (+)

Lane 3:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab208589 in HepG2 whole cell lysate (-)

Secondary

All lanes:

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

Observed band size: 33-72 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Prothrombin antibody [EPR20159] (AB208589)

Blocking/Dilution buffer : 5% NFDM/TBST.

Prothrombin is proteolytically cleaved into different fragments. The fragments profile is consistent with what has been described in the literature (PMID : 17637839; PMID : 21131592; PMID : 16734589).

All lanes:

Western blot - Anti-Prothrombin antibody [EPR20159] (ab208589) at 1/1000 dilution

Lane 1:

Human plasma at 20 µg

Lane 2:

Human fetal liver tissue lysate at 20 µg

Lane 3:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Predicted band size: 70 kDa

Observed band size: 33 kDa,52 kDa,72 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Prothrombin antibody [EPR20159] (AB208589)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Low expression : SW480 (PMID : 31324782). ab314480 demonstrated significant selectivity for a-thrombin over prothrombin. This data is displayed compared to ab208589 which demonstrates enhanced selectivity for prothrombin over a-thrombin. The expression profile/molecular weight observed is consistent with what has been described in the literature (PMID : 31324782, 21527525, 22345489). Lanes 1-3 are applied with Anti-Thrombin antibody [EPR25304] (ab314480) at 1/1000 dilution and lanes 4-6 are applied with Anti-Prothrombin antibody [EPR20159] (ab208589) at 1/1000 dilution. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Lanes 1 - 3:

Western blot - Anti-Thrombin antibody [EPR25304-12] (<a href='/ko/products/primary-antibodies/thrombin-antibody-epr25304-12-ab314480'>ab314480</a>) at 1/1000 dilution

Lanes 4 - 6:

Western blot - Anti-Prothrombin antibody [EPR20159] (ab208589) at 1/1000 dilution

Lanes 1 and 4:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 2 and 5:

Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 3 and 6:

SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 38 kDa,37 kDa

false

Exposure time: 59s