All lanes : Anti-AKT1 antibody [M1843] (ab108202) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : AKT1 knockout HeLa cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : Daudi cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 55 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Lanes 1- 4: Merged signal (red and green). Green - ab108202 observed at 60 kDa. Red - Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) observed at 37 kDa.

 ab108202 was shown to react with AKT1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264852 (knockout cell lysate ab256835) was used. Wild-type HeLa and AKT1 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. ab108202 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Allele-1: 1 bp insertion in exon 2.

 

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

 

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