All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/10000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 58 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab150358 observed at 160 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab150358 was shown to react with PODXL in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264984 (knockout cell lysate ab257210) was used. Wild-type HeLa and PODXL 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. ab150358 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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.

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.