Overlay histogram showing HepG2 cells stained with ab137123 (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 (ab137123, 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 HepG2 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-alpha 1a Adrenergic Receptor/ADRA1A antibody [EPR9691(B)] (ab137123) at 1/1000 dilution

Lane 1 : PC-3 cell lysate
Lane 2 : HepG2 cell lysate
Lane 3 : Raji cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 60 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?

Immunofluorescent analysis of HepG2 cells labelling alpha 1a Adrenergic Receptor/ADRA1A with ab137123 at 1/250 dilution.