Recombinant Alexa Fluor® 647 Anti-ERK2 antibody [E460] (ab190581)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-MAPK1 knockout cells (red line) stained with ab190581. 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 (ab190581, 0.01µg/ml dilution) for 30 min at 22°C.

A rabbit monoclonal IgG isotype control antibody  (ab199093) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-MAPK1   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 40 mW Red laser (640nm) and 670/14 bandpass filter.

Overlay histogram showing HeLa cells stained with ab190581 (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 (ab190581, 1/500 dilution) for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor® 647 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 solid-state 25mW red diode laser (635 nm) and 675/30 bandpass filter.

This antibody gave a positive signal in HeLa fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.