Primary Antibodies: Lane 1: Anti Akt-1 antibody ( ab54752 ) at 5?g/mL dilution. Lane 2: Anti Akt-1 antibody ( ab81283 ) at 1/5000 dilution. Sample: Akt-1 recombinant protein (Tagged)(ab62279) ? 50ng. Secondary Antibody: Lane 1: Goat polyclonal to Mouse IgG ? H&L ? Pre-Adsorbed (HRP) at 1:5000 developed using the ECL technique. Lane 2: Goat polyclonal to Rabbit IgG ? H&L - Pre-Adsorbed (HRP) at 1:5000 developed using the ECL technique. Performed under reducing conditions. Predicted band size : 80kDa. Observed band size : 80kDa. Blocking step: 5% Milk in 50mM TBS+0.05% Tween for 1 hour at RT. Primary antibody buffer: 5% BSA in 50mM TBS+0.05% Tween overnight at 4?C. Secondary antibody buffer: 10% blocking ab126587 in water for 2 hours at RT. Exposure time : 5 minutes