All lanes : Anti-ROCK2 antibody [EPR7141(B)] (ab125025) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ROCK2 knockout HeLa cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 161 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 - ab125025 observed at 175 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab125025 was shown to react with ROCK2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265679 (knockout cell lysate ab257643) was used. Wild-type HeLa and ROCK2 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. ab125025 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: 59 bp deletion in exon 4.

 

Allele-2: 1 bp deletion in exon 4.