Overlay histogram showing Raji cells stained with ab92313 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab92313, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Raji cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
Western blot - Anti-RHEB antibody [EPR2971] (ab92313)
All lanes : Anti-RHEB antibody [EPR2971] (ab92313) at 1/2000 dilution
Lane 1 : Raji cell lsyate Lane 2 : SH-SY5Y cell lsyate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution