Recombinant Alexa Fluor® 647 Anti-MEK1 antibody [E342] (ab193986)

ab193986 staining MEK1 in HeLa cells. The cells were fixed with 100% methanol (10 min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Triton X-100 for 1hr. The cells were then incubated with ab193986 at a working dilution of 1 in 50 (shown in red) and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin) at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1hr with an AlexaFluor^® 488 Goat anti-mouse IgG (H&L - preadsorbed) secondary (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

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

Image was taken with a Confocal microscope (Leica micro-systems, TCS SP8).

Overlay histogram showing HeLa cells stained with ab193986 (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 (ab193986, 0.2μl/1x10⁶ cells) for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (Alexa Fluor^® 647) (0.1μg/1x10⁶ cells) for 30 min at 22ºC. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 25mW red solid state diode laser (635nm) 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.