All lanes : Anti-Factor H antibody [EPR6225] (ab133536) at 1/1000 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : CFH knockout A549 cell lysate
Lane 3 : HaCaT 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: 139 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab133536 observed at 180 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab133536 Anti-Factor H antibody [EPR6225] was shown to specifically react with Factor H in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267031 (knockout cell lysate ab257150) was used. Wild-type and Factor H knockout samples were subjected to SDS-PAGE. ab133536 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: 10 bp deletion in exon3

 

Allele-2: 2 bp deletion in exon 3.

 

Allele-3: 1 bp deletion in exon 3.

 

Representative images of CFH 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 an EVOS M5000 microscope.