Non radioactive akt kinase assay kit

A total of 500 μg protein was incubated with anti-Akt antibody and Sepharose beads overnight at 4 °C. After binding, the beads were washed four times with lysis buffer. Akt activity was measured using a non-radioactive Akt kinase assay kit (Cell Signaling Technology, Inc., Beverly, MA, USA).

After appropriate treatments, Akt kinase assay was performed using a non-radioactive Akt kinase assay kit (Cell Signaling Technology) following the manufacturer’s instructions. Briefly, Akt was immunoprecipitated from 100 μg cell lysate overnight using an immobilized Akt antibody. The beads were then pelleted and washed twice in 500 μL of cell lysis buffer and twice in 500 μL of kinase buffer. Pellets were resuspended in 25 μL of kinase buffer containing 0.5 mL of 10 mM ATP, 1 mg Glycogen synthase kinase-3 (GSK-3) fusion protein for 30 min at 30 °C. Kinase reaction was terminated by adding sample buffer containing β-mercapatoethanol. Finally, the activity of Akt kinase in each sample was determined according to GSK-3α/β phosphorylation by Western blot.