Supersignal westfemto ecl reagent

Cell lysates (7 μg protein/sample) were diluted in SDS sample buffer (0.31 M Tris-HCl, pH 6.8, 50% glycerol, 10% SDS, 100 mM DTT, 0.01% bromophenol blue, and 1 M β-mercaptoethanol) and incubated at 65 °C for 10 min with shaking. Proteins were separated via electrophoresis on 7% SDS-polyacrylamide gel in Laemmli buffer and electro-transferred in Towbin buffer onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% non-fat dried milk in PBS for 60 min, at RT. Pgp was labeled with D3H1Q (Cell Signaling Technology) human anti-Pgp rabbit monoclonal primary antibody (1:1000 diluted in 5% non-fat dried milk in PBS) for 60 min, at RT with continuous agitation. Non-bound antibodies were washed with PBS containing 0.1% Tween-20 3 times for 10 min, at RT with continuous shaking. They were then labeled with anti-rabbit goat IgG secondary antibody conjugated with horseradish peroxidase (diluted in a ratio of 1:5000 in 5% non-fat dried milk in PBS) for 60 min, at RT with continuous shaking. The unbound antibodies were washed 3 times, then the immunoblots were developed with SuperSignal West Femto ECL reagent (Thermo Fisher Scientific) and images were recorded using the Chemidoc imaging system (Bio-Rad Hungary Ltd., Budapest, Hungary).