All lanes : Anti-EEA1 antibody [EPR4245] - Early Endosome Marker (ab109110) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : EEA1 knockout HeLa cell lysate
Lane 3 : Daudi cell lysate
Lane 4 : SH-SY5Y cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 162 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?

Lanes 1- 4: Merged signal (red and green). Green - ab109110 observed at 175 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109110 was shown to react with EEA1 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab261822 (knockout cell lysate ab256897) lane below 175kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and EEA1 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. ab109110 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: 1 bp insertion in exon 13.