Anti-BCAT2 antibody [EPR27488-72]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-BCAT2 antibody [EPR27488-72] (AB307833)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (human breast adenocarcinoma epithelial cell) cells labelling BCAT2 with ab307833 at 1/500 dilution (1.1 ug/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green). Confocal image showing mitochondrial staining in MCF7 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab197491 Anti-COX IV Mouse monoclonal antibody (Alexa Fluor® 647) - Mitochondrial Marker was used to counterstain tubulin at 1/200 dilution (2.5 ug/ml)(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 dilution (2 ug/ml).

• IP

Supplier Data

Immunoprecipitation - Anti-BCAT2 antibody [EPR27488-72] (AB307833)

BCAT2 was immunoprecipitated from 0.35 mg MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate with ab307833 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307833 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : MCF7 whole cell lysate 10 ug Lane 2 : ab307833 IP in MCF7 whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307833 in MCF7 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : Lanes 1-3 : 6 seconds (left), Lanes 1-3 : 180 seconds (right). The IP experiment was performed by ab307833 using MCF cells. On the left the IP blot was probed with ab307833 and on the right the blot was probed by another anti-BCAT2 antibody (ab95976)(1 : 1000 dilution).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-BCAT2 antibody [EPR27488-72] (AB307833)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 mouse embryonic fibroblast cells labelling BCAT2 with ab307833 at 1/500 dilution (0.1ug) (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-BCAT2 antibody [EPR27488-72] (AB307833)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling BCAT2 with ab307833 at 1/500 dilution (1.1 ug/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green). Confocal image showing cytoplasmic staining in NIH/3T3 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 ug/ml) (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 dilution (2 ug/ml).

• IP

Supplier Data

Immunoprecipitation - Anti-BCAT2 antibody [EPR27488-72] (AB307833)

BCAT2 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab307833 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307833 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : NIH/3T3 whole cell lysate 10 ug Lane 2 : ab307833 IP in NIH/3T3 whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307833 in NIH/3T3 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 3 seconds.

• WB

Supplier Data

Western blot - Anti-BCAT2 antibody [EPR27488-72] (AB307833)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Exposure time : Lane 1, 4-5 : 15 seconds, Lane 2-3 : 81 seconds.

All lanes:

Western blot - Anti-BCAT2 antibody [EPR27488-72] (ab307833) at 1/1000 dilution

Lane 1:

Human skeletal muscle tissue lysate at 20 µg

Lane 2:

Human liver tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Mouse heart tissue lysate at 20 µg

Lane 5:

Mouse skeletal muscle tissue 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: 37 kDa

false

• WB

Supplier Data

Western blot - Anti-BCAT2 antibody [EPR27488-72] (AB307833)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. This antibody does not cross-react with human BCAT1. In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution. In Western blot, anti-H3 antibody (ab176842) loading control staining at 1/100000 dilution. Exposure time : 1 second.

All lanes:

Western blot - Anti-BCAT2 antibody [EPR27488-72] (ab307833) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell)cells transfected with an empty vector containing a his tag whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human BCAT2 expression vector containing a his tag whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human BCAT1 expression vector containing a his tag 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 1/20000 dilution

Predicted band size: 44 kDa

Observed band size: 37 kDa

false

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-BCAT2 antibody [EPR27488-72] (AB307833)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 15 seconds.

All lanes:

Western blot - Anti-BCAT2 antibody [EPR27488-72] (ab307833) at 1/1000 dilution

Lane 1:

Mouse stomach tissue lysate at 20 µg

Lane 2:

Human stomach tissue lysate at 20 µg

Lane 3:

Rat stomach tissue 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: 37 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-BCAT2 antibody [EPR27488-72] (AB307833)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution. Exposure time : 15 seconds.

All lanes:

Western blot - Anti-BCAT2 antibody [EPR27488-72] (ab307833) at 1/1000 dilution

Lane 1:

MCF7 whole cell lysate at 20 µg

Lane 2:

Jurkat whole cell lysate at 20 µg

Lane 3:

Raji whole cell lysate at 20 µg

Lane 4:

HeLa whole cell lysate at 20 µg

Lane 5:

NIH/3T3 whole cell lysate at 20 µg

Lane 6:

RAW264.7 whole cell lysate at 20 µg

Lane 7:

PC-12 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: 37 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-BCAT2 antibody [EPR27488-72] (AB307833)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. The expression of BCAT2 is upregulated in response to 3T3-L1 differentiation for 7 days treatment (PMID : 27689828). In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution. Exposure time : 6 seconds.

All lanes:

Western blot - Anti-BCAT2 antibody [EPR27488-72] (ab307833) at 1/1000 dilution

Lane 1:

Untreated 3T3-L1 whole cell lysate at 20 µg

Lane 2:

3T3-L1 differentiation for 7 days 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 1/20000 dilution

Observed band size: 37 kDa

false

Exposure time: 6s