All lanes : Anti-PKN antibody [EPR3237] (ab108976) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : PKN1 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate
Lane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole 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: 104 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab108976 observed at 120 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab108976 Anti-PKN antibody [EPR3237] was shown to specifically react with PKN in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266599 (knockout cell lysate ab258586) was used. Wild-type and PKN knockout samples were subjected to SDS-PAGE. ab108976 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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: 1 bp insertion in exon2

 

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