Jurkat cells (growing in DMEM supplemented with 10% serum) were washed, serum starved (0% Serum) for 3 hours. 20% serum were then added to the culture for 30 minutes to stimulate the cells. The serum-activated cells were washed with ice-cold PBS, then lyzed in three volume of Kinase Extraction Buffer (70002-N80M in K435-40), keep on ice for 5 min, vortex, centrifuge at top speed for 15 min, collect the cell lysate. Extract the pellet one more time with one volume extraction buffer. Combine the cell lysate together. Determine protein concentration. The cell lysate contains activated Akt, which can be used as a positive control in Akt activity assay, in conjunction with the use of BioVision's Akt Activity Assay Kit (K435-40). We recommend usi