All lanes : Anti-CD146 antibody [EPR3208] (ab75769) at 1/1000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab75769 observed at 120 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab75769 was shown to react with CD146 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab261790 (knockout cell lysate ab256985) lane below 120kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and MCAM 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. ab75769 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated 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: 19 bp deletion in exon 6.