All lanes : Anti-Cytochrome P450 Reductase antibody [EPR14479(B)] (ab180597) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : POR knockout HCT116 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Predicted band size: 76 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Lanes 1- 4: Merged signal (red and green). Green - ab180597 observed at 80 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab180597 was shown to react with Cytochrome P450 Reductase in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266889 (knockout cell lysate ab257596) was used. Wild-type HCT116 and POR knockout HCT116 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. ab180597 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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.

Homozygous: Insertion of the selection cassette in exon4