ICC/IF image of ab45295 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab45295, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (5 min) HeLa cells at 5µg/ml.

Anti-PGE2 receptor EP4 subtype antibody (ab45295) at 1 µg/ml + Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 20 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 53 kDa
Observed band size: 73 kDa why is the actual band size different from the predicted?
Additional bands at: 30 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes


ab45295 detects a 73 kDa band in Western Blot which we believe corresponds to the glycosylated form of the protein.