Protein Kinase B Alpha (PKBa), also known as AKT1, is a key member of the AKT family of serine/threonine kinases, which play a crucial role in various cellular processes including cell growth, proliferation, differentiation, and survival. PKBa is activated by insulin and growth factor signaling and phosphorylates a variety of substrates to regulate these cellular functions.The activation of akt1 requires the association with Heat Shock Protein 90kDa Alpha B1 (HSP90aB1), resulting in an active auto-regulatory cycle.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human PKBa and recombinant human HSP90aB1. Briefly, PKBa was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to HSP90aB1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-PKBa pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 μL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant human PKBa and recombinant human HSP90aB1 was shown in Figure 1, the EC50?for this effect is 0.149ug/mL.