All lanes : Anti-hHR23A antibody [EPR4817] (ab108591) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : RAD23A knockout HEK293T cell lysate
Lane 3 : MCF7 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: 40 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab108591 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab108591 Anti-hHR23A antibody [EPR4817]  was shown to specifically react with hHR23A in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266343 (knockout cell lysate ab258163) was used. Wild-type and hHR23A knockout samples were subjected to SDS-PAGE. ab108591 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C 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: 2 bp deletion in exon2

 

Allele-2: 1 bp deletion in exon 2.

 

Representative images of RAD23A 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.