Overlay histogram showing HAP1 wildtype (green line) and HAP1-EIF4EBP1   knockout cells (red line) stained with ab213659. 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 (ab213659, 0.1µg/ml dilution) for 30 min at 22°C.

A rabbit monoclonal IgG isotype control antibody  (ab209478) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-EIF4EBP1  knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

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

This antibody can also be used in HAP1 cells fixed with 4% formaldehyde (10 min) , permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

Overlay histogram showing HeLa cells stained with ab213659 (red line). The cells were fixed with 4% formaldehyde (10 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 (ab213659, 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.

This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.