All lanes : Anti-BRD3 antibody [2088C3a] (ab50818) at 1/500 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : BRD3 knockout HEK293T cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution

Predicted band size: 80 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab50818 observed at 100 kDa. Red - loading control ab181602 observed at 36 kDa.

 ab50818 Anti-BRD3 antibody [2088C3a] was shown to specifically react with BRD3 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266793 (knockout cell lysate ab258335) was used. Wild-type and BRD3 knockout samples were subjected to SDS-PAGE. ab50818 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Homozygous: 1 bp deletion in exon 4

Representative images of BRD3 knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using an EVOS M5000 microscope.