Recombinant Anti-Brd4 antibody [EPR5150(2)] (ab128874)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Brd4 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab128874 observed at 150 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab128874 was shown to recognize Brd4 when Brd4 knockout samples were used, along with additional cross-reactive bands. Wild-type and Brd4 knockout samples were subjected to SDS-PAGE. ab128874 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Unpurified ab128874 (1/500) staining Brd4 in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells (green). Cells were fixed in paraformaldehyde, permeabilized in 0.5% Triton X100/PBS and counterstained with DAPI in order to highlight the nucleus (red).

For further experimental details please refer to Abreview.

See Abreview

Unpurified ab128874 at 1/100 dilution staining Brd4 in human colon carcinoma tissue by Immunohistochemistry.

Flow cytometry analysis of SW480 (Human colorectal adenocarcinoma epithelial cell) cells labeling Brd4 (red) with purified ab128874 at a 1/50 dilution (10 µg/mL). Cells were fixed with 80% methanol and permeabilized with 0.1% Tween-20. A goat anti rabbit IgG (Alexa Fluor^®488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue).

ab128874 at 1000 µg immunoprecipitating Brd4 in HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate. Sample was incubated with unpurified primary antibody (50 mM Tris dilution buffer) and protein G for 16 hours at 4°C.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

See Abreview

Immunocytochemistry/Immunofluorescence analysis of HepG2 (Human liver cell line) cells labeling Brd4 with purified ab128874 at 1/100 (Panel A). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain(Panel B).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue labeling Brd4 with purified ab128874 at 1/200. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. An undiluted Goat Anti-Rabbit IgG H&L (HRP)  was used as the secondary antibody at. Counterstained with hematoxylin.

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

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