All lanes : Anti-APPL antibody [EPR13569] (ab180140) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : APPL knockout HeLa cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 80 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Lanes 1- 4: Merged signal (red and green). Green - ab180140 observed at 90 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab180140 was shown to react with Anti-APPL in wild-type HeLa cells in western blot. The band observed in knockout cell line ab265187 (knockout cell lysate ab257836) lane below 90kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and APPL1 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. ab180140 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 10000 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: 2 bp deletion in exon 2.