All lanes : Anti-PDCD6/ALG-2 antibody [EP3259] (ab109181) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : PDCD6 knockout HEK293T cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Daudi 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: 21 kDa
Observed band size: 22 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab109181 observed at 22 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab109181 Anti-PDCD6/ALG-2 antibody [EP3259] was shown to specifically react with PDCD6/ALG-2 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266323 (knockout cell lysate ab258570) was used. Wild-type and PDCD6/ALG-2 knockout samples were subjected to SDS-PAGE. ab109181 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.

Homozygous: 10 bp deletion in exon 1

Representative images of PDCD6 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.