All lanes : Anti-ATF6 antibody (ab83504) at 1 µg/ml

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ATF6 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 74 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab83504 observed at 95 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab83504 was shown to react with ATF6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261800 (knockout cell lysate ab256841) was used. Wild-type HeLa and ATF6 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. ab83504 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at 1 µg/ml 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: 25 bp deletion in exon 6.

 

Allele-2: 17 bp deletion in exon 6.

 

Allele-3: 4 bp deletion in exon 6.