Jurkat cells growing in DMEM supplemented with 10% FBS were collected and washed in ice-cold PBS. Cells were then lyzed in three volume of Kinase Extraction Buffer (70002-N80M in K435-40), keep on ice for 5 min, centrifuge at top speed for 15 min. Collect the cell lysate. Extract the pellet again with one volume Kinase Extraction Buffer. Combine the cell lysate together, determine protein concentration. The cell lysate can be used as a negative control for Akt activity assays, in conjunction with the use of BioVision’s Akt Activity Assay Kit (K435-40). We recommend using 100 µg per assay.