Phospho akt antibody

Huh7 cells (200 000 per well) were seeded in 4 mL medium using six-well plates. After 24 h, medium was replaced with 2 mL fresh medium. The transfections were performed with polyplexes containing 5 µg pDNA in a total volume of 500 μL. After 45 min of incubation, the cells were lysed and total protein concentration was determined using a BCA assay. Equal amounts of protein (30 μg) in loading buffer were applied per lane and were separated by SDS-PAGE under reducing conditions, blotted on nitrocellulose membrane and blocked with NET gelatine for 1 h at room temperature. Immunostaining was performed using Met (Santa Cruz Biotechnology, USA), phospho-Met (Cell Signalling, USA), Akt and phospho-Akt antibodies (Cell Signaling, Germany) overnight at 4 °C. After the incubation with the applicable primary antibodies, membranes were washed three times for 15 min with NET gelatine before incubating with the adequate secondary peroxidase antibody for 1 h. When necessary the membranes were stripped in 2% SDS (w/v) with 0.8% (v/v) β-mercaptoethanol in 0.07 M Tris/HCl ( pH 6.8) solution for 1 h at 50 °C. After another three washing cycles, the membranes were cut accordingly and the proteins were then visualized using Lumi-Light Western blotting substrate (Roche, Germany).