Overlay histogram showing HeLa cells stained with ab210454 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 90% methanol for 30 min at -20°C. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab210454, 1/2500 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 50mW Yellow-Green laser (561nm) and 586/15 bandpass filter.

ab210454 staining AKT1 + AKT2 + AKT3 in HeLa cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab210454 at 1/500 dilution (pseudocolored in green) and ab195884, Rat monoclonal to Tubulin (Alexa Fluor^® 647), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in HeLa cells fixed with 4% formaldehyde (10 min).