All lanes : Anti-CD147 antibody [EPR4053] (ab108308) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : BSG knockout HEK293T cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : U-87 MG 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

Performed under reducing conditions.

Predicted band size: 42 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab108308 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa.

ab108308 Anti-CD147 antibody [EPR4053] was shown to specifically react with CD147 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266331 (knockout cell lysate ab256853) was used. Wild-type and CD147 knockout samples were subjected to SDS-PAGE. ab108308 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.

Allele-1: 5 bp deletion in exon 4

 

Allele-2: 44 bp insertion in exon 4.

 

Representative images of BSG 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.