All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : PODXL knockout HCT116 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 58 kDa
Observed band size: 200 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab150358 observed at 200 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab150358 was shown to react with PODXL in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266887 (knockout cell lysate ab257211) was used. Wild-Type HCT116 and PODXL 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. ab150358 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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: 1 bp insertion in exon2

Representative images PODXL knockout HCT116 cells, low and high confluency examples (top left and right respectively) and wild-type HCT116 cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS M5000 microscope.