All lanes : Anti-E2F4 antibody [EPR8259] (ab150360) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : E2F4 knockout HEK293T cell lysate
Lane 3 : K-562 cell lysate
Lane 4 : Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 44 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab150360 observed at 60 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab150360 Anti-E2F4 antibody [EPR8259] was shown to specifically react with E2F4 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266119 (knockout cell lysate ab257932) was used. Wild-type and E2F4 knockout samples were subjected to SDS-PAGE. ab150360 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

Allele-1: 4 bp deletion in exon2

 

Allele-2: Insertion of the selection cassette in exon 2.