Anti-Brd4 antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody (AB75898)

ICC/IF image of ab75898 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab75898, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa, Hek293 and HepG2 cells at 5µg/ml.

• WB

Unknown

Western blot - Anti-Brd4 antibody (AB75898)

All lanes:

Western blot - Anti-Brd4 antibody (ab75898) at 1 µg/mL

Lane 1:

Liver (Mouse) Tissue Lysate at 10 µg/mL

Lane 2:

Liver (Rat) Tissue Lysate at 10 µg/mL

Lane 3:

Human liver tissue lysate - total protein (<a href='/en-us/products/unavailable/human-liver-tissue-lysate-total-protein-ab29889'>ab29889</a>) at 10 µg/mL

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP)

Predicted band size: 152 kDa

Observed band size: 171 kDa,70 kDa

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Exposure time: 150s

• WB

Lab

Western blot - Anti-Brd4 antibody (AB75898)

Lanes 1 - 2 : Merged signal (red and green). Green - ab75898 observed at 220 kDa. Red - loading control Mouse anti Vinculin observed at 125 kDa.

ab75898 was shown to react with Brd4 in HAP1 wild-type cells in Western blot with loss of signal observed in BRD4 knockout sample. Wild-type HAP1 and BRD4 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab75898 and Mouse anti Vinculin overnight at 4 °C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Brd4 antibody (ab75898) at 1 µg/mL

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

BRD4 knockout HAP1 cell lysate at 20 µg

Predicted band size: 152 kDa

Observed band size: 220 kDa

false

• ICC/IF

AbReview27967****

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody (AB75898)

ab75898 (1/500) staining Brd4 in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

Image courtesy of an Abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada