All lanes : Anti-TRIM21/SS-A antibody [EPR20290] (ab207728) at 1/500 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : TRIM21 knockout A549 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : MOLT-4 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: 54 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 - ab207728 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab207728 Anti-TRIM21/SS-A antibody [EPR20290] was shown to specifically react with TRIM21/SS-A in wild-type A549 cells. Loss of signal was observed when knockout cell line ab267025 (knockout cell lysate ab257767) was used. Wild-type and TRIM21/SS-A knockout samples were subjected to SDS-PAGE. ab207728 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.

Allele-1: 13 bp deletion in exon4

 

Allele-2: 1 bp insertion in exon 4.

 

Representative images of TRIM21 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.