ICC/IF image of ab31533 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab31533, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Anti-Isoleucyl tRNA synthetase antibody (ab31533) at 1/2000 dilution + whole cell lysate prepared from HEK293 cells at 50 µg/ml

Secondary
Goat anti-rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 145 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?