Overlay histogram showing HeLa cells stained with ab2781 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2781, 2µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

All lanes : Anti-NCOR2/SMRT antibody [1542] (ab2781) at 1/100 dilution

Lane 1 : HeLa cell lysate
Lane 2 : Neuro-2a cell lysate

Lysates/proteins at 25 µg per lane.

Predicted band size: 274 kDa
Observed band size: 220,250 kDa why is the actual band size different from the predicted?