All lanes : Anti-Glycerol kinase antibody [EPR6567] (ab126599) at 1/500 dilution

Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : GK knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : HepG2 (Human liver hepatocellular carcinoma cell line) 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: 61 kDa
Observed band size: 61 kDa

Lanes 1-4: Merged signal (red and green). Green - ab126599 observed at 61 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab126599 Anti-Glycerol kinase antibody [EPR6567] was shown to specifically react with Glycerol kinase in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab267328 (knockout cell lysate ab257966) was used. Wild-type and Glycerol kinase knockout samples were subjected to SDS-PAGE. ab126599 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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^® 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: 1 bp insertion in exon4

Representative images of GK 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 a EVOS M5000 microscope.