All lanes : Anti-VRK1 antibody [5D1] - N-terminal (ab171933) at 1/500 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : VRK1 knockout HEK293T cell lysate
Lane 3 : 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: 45 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab171933 observed at 50 kDa. Red - loading control ab181602 observed at 36 kDa.

 ab171933 Anti-VRK1 antibody [5D1] - N-terminal was shown to specifically react with VRK1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266257 (knockout cell lysate ab258283) was used. Wild-type and VRK1 knockout samples were subjected to SDS-PAGE. ab171933 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: 20 bp deletion in exon 2

Representative images of VRK1 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.