Overlay histogram showing HeLa cells stained with ab224972 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab224972, 1/1000 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

All lanes : Anti-ROCK2 + ROCK1 antibody [EP786Y] (ab45171) at 1/1000 dilution

All lanes : Recombinant Human ROCK2 protein (aa 1132 to 1388) (30 kDa)

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 30 kDa why is the actual band size different from the predicted?

This data was developed using ab45171, the same antibody clone in a different buffer formulation.

Loading control: Anti-6X His tag® antibody [EPR20547] (ab213204)

Blocking buffer and concentration: 5% NFDM/TBST

Exposure Times:

Lane 1: 3 seconds

Lane 2: 7 seconds