Recombinant Anti-BRD3 antibody [EPR23743-226] (ab300106)

All lanes : Anti-BRD3 antibody [EPR23743-226] (ab300106) at 1/1000 dilution

Lane 1 : Wild type HEK-293T (human embryonic kidney epithelial cell), whole cell lysate
Lane 2 : BRD3 knockout HEK-293T whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at 1/10000 dilution

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

Blocking and dilution buffer and concentration: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS. 

The identity of the lower MW band at approximately 50kDa is unknown.
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
False colour image of Western blot: Anti-BRD3 antibody [EPR23743-226] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab300106 was shown to bind specifically to BRD3. A band was observed at 100 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in BRD3 knockout cell line ab266793 (knockout cell lysate ab258335). To generate this image, wild-type and BRD3 knockout HEK-293 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/10000 dilution.