All lanes : Anti-galectin 9/Gal-9 antibody [EPR22214] (ab227046) at 1/500 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : LGALS9 knockout A549 cell lysate
Lane 3 : U-937 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: 34-39 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab227046 observed at 34-39 kDa. Red - loading control ab8245 observed at 36 kDa.

ab227046 Anti-galectin 9/Gal-9 antibody [EPR22214] was shown to specifically react with galectin 9/Gal-9 in wild-type A549 cells. Loss of signal was observed when knockout cell line ab266923 (knockout cell lysate ab256976) was used. Wild-type and galectin 9/Gal-9 knockout samples were subjected to SDS-PAGE. ab227046 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 exon3

Representative images of Lgals9 knockout A549 cells, low and high confluency examples (top left and right respectively) and wild-type A549 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 XL Core microscope.