All lanes : Anti-DAZAP1 antibody [EPR14400-60] (ab184183) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : DAZAP1 knockout HEK293T cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HeLa 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: 43 kDa
Observed band size: 43 kDa

Lanes 1-4: Merged signal (red and green). Green - ab184183 observed at 43 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab184183 Anti-DAZAP1 antibody [EPR14400-60]  was shown to specifically react with DAZAP1 in wild-type HEK293T cells. The band observed in knockout cell line ab266469 (knockout cell lysate ab257911) lane below 43 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and DAZAP1 knockout samples were subjected to SDS-PAGE. ab184183 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: 40 bp deletion in exon 6

 

Allele-2: Insertion of the selection cassette in exon 6.

 

Representative images of DAZAP1 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.