Active Glycogen Synthase Kinase 3 Beta (GSK3b)

Glycogen synthase kinase 3β (GSK3β) is a multifunctional protein kinase that targets Ser/Thr residues with a priming phosphorylation at the fourth amino acid on the C-terminal side. GSK3β is ubiquitously expressed in all types of cells and tissues and is particularly abundant in the brain. GSK3β regulates many cellular functions, including cell proliferation, cell survival, gene expression, cellular architecture, neural development and plasticity etc. Besides, Adenomatosis Polyposis Coli Protein (APC) can bind to GSK3β to form complexes with specific functions. This complex plays a central role in the Wnt signaling pathway, which is involved in regulating the stability of β-catenin, thus a functional ELISA assay was conducted to detect the interaction of recombinant human GSK3b and recombinant mouse APC. Briefly, GSK3b was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to APC-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-GSK3b 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 450/630 nm immediately. The binding activity of recombinant human GSK3b and recombinant mouse APC was shown in Figure 1, the EC50 for this effect is 0.34 ug/mL.