All lanes : Anti-PAK1 antibody [EPR20048] (ab223849) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PAK1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 61 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab223849 observed at 65 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab223849 was shown to react with PAK1 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab264889 (knockout cell lysate ab257572) lane below 65kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and PAK1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab223849 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 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 2.

 

Allele-2: 2 bp deletion in exon 2.

 

Allele-3: Insertion of the selection cassette in exon 2.