Anti-BARD1 antibody [EPR29601-553]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-BARD1 antibody [EPR29601-553] (AB324523)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) cells labelling BARD1 with ab324523 at 1/50 (10.2 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2ug/ml) dilution (Green).

Confocal image showing mainly nuclear staining in 293T cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Low expression : SK-OV-3

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1ug/ml) dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab324523 at 1/50 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution.

-ve control 2 : ab7291 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-BARD1 antibody [EPR29601-553] (AB324523)

BARD1 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab324523 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab324523 at 1000 dilution.

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

To minimize protein degradation, cells were lysed immediately after harvest and then applied for Immunoprecipitation as soon as possible.

All lanes:

Immunoprecipitation - Anti-BARD1 antibody [EPR29601-553] (ab324523) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate(input) at 10 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate(+)

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab324523 in HeLa 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

Observed band size: 87 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-BARD1 antibody [EPR29601-553] (AB324523)

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

Low expression : SK-OV-3 (PMID : 18089818).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-BARD1 antibody [EPR29601-553] (ab324523) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate at 50 µg

Lane 2:

SK-OV-3 (human ovarian cancer epithelial cell) whole cell lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 87 kDa,36 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-BARD1 antibody [EPR29601-553] (AB324523)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-BARD1 antibody [EPR29601-553] (ab324523) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 50 µg

Lane 2:

HeLa transfected with siRNA specifically targeting BARD1 whole cell lysate at 50 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: 87 kDa,36 kDa

false

Exposure time: 8s