All lanes : Anti-Heparanase 1 antibody [EPR22365-230] (ab254254) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : HPSE knockout HeLa cell lysate
Lane 3 : Capan-1 cell lysate
Lane 4 : Human pancreas tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 61 kDa
Observed band size: 52,65 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab254254 observed at 52,65 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab254254 Anti-Heparanase 1 antibody [EPR22365-230] was shown to specifically react with Heparanase 1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265413 (knockout cell lysate ab258456) was used. Wild-type and Heparanase 1 knockout samples were subjected to SDS-PAGE. ab254254 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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: 17 bp deletion in exon 1.