Anti-Brd4 antibody [EPR25425-23]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Brd4 antibody [EPR25425-23] (AB289886)

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling Brd4 with ab289886 at 1/500 dilution (0.1μg) (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 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR25425-23] (AB289886)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labeling Brd4 with ab289886 at 1/50 dilution, followed by ab150081 Goat Anti-Rbit IgG H&L (Alexa Fluo^r® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 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 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR25425-23] (AB289886)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labelling Brd4 with ab289886 at 1/2000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nucleus staining was found in rat testis. The section was incubated with ab289886 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR25425-23] (AB289886)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labelling Brd4 with ab289886 at 1/2000 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining was found in mouse testis (PMID : 28076398). The section was incubated with ab289886 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BON^D® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Brd4 antibody [EPR25425-23] (AB289886)

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling Brd4 with ab289886 at 1/500 dilution (0.1μg) (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 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR25425-23] (AB289886)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labeling Brd4 with ab289886 at 1/50 dilution, followed by ab150081 Goat Anti-Rbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubμle Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 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 dilution.

• WB

Supplier Data

Western blot - Anti-Brd4 antibody [EPR25425-23] (AB289886)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-Brd4L antibody [EPR25425-23] (ab289886) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-pan Brd4 antibody [EPR25424-71] (<a href='/en-us/products/primary-antibodies/pan-brd4-antibody-epr25424-71-ab314432'>ab314432</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

293T transfected with siRNA specifically targeti Brd4 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: 110 kDa,200 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-Brd4 antibody [EPR25425-23] (AB289886)

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

Lysates were prepared from fresh material and used for Western blotting immediately to minimize protein degradation.

Exposure time : 48 seconds.

All lanes:

Western blot - Anti-Brd4 antibody [EPR25425-23] (ab289886) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 152 kDa

Observed band size: 200 kDa

false

• WB

Lab

Western blot - Anti-Brd4 antibody [EPR25425-23] (AB289886)

Western blot : Rabbit Monoclonal [EPR25425-23] to Brd4 ab289886 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 200 kDa. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Brd4 antibody [EPR25425-23] (ab289886) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (whole lysate) at 20 µg

Lane 2:

Wild-type HAP1 (Nuclear fraction) at 20 µg

Lane 3:

Wild-type HAP1 (Membrane fraction) at 20 µg

Lane 4:

HEK-293T (Whole lysate) at 20 µg

Lane 5:

HeLa (Whole lysate) at 20 µg

Lane 6:

HeLa (Nuclear fraction) at 20 µg

Lane 7:

HeLa (Membrane fraction) at 20 µg

Secondary

Lanes 1 - 5:

Goat anti-Rabbit 800CW at 1/20000 dilution

Lanes 1 - 5:

Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 200 kDa

Observed band size: 200 kDa,50 kDa

false

• WB

Lab

Western blot - Anti-Brd4 antibody [EPR25425-23] (AB289886)

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

This blot was developed using a higher sensitivity ECL substrate.

Exposure time : 180 seconds

All lanes:

Western blot - Anti-Brd4 antibody [EPR25425-23] (ab289886) at 1/1000 dilution

Lane 1:

Mouse testis tissue lysate at 20 µg

Lane 2:

Rat testis tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 152 kDa

Observed band size: 200 kDa

false