Determination of Kd value using Alpha assay

Alpha binding assay using Streptavidin Donor beads and anti-His6 AlphaLISA^® Acceptor Beads (PerkinElmer) was used to determine K_d for the PTEN: PIP_3, or PTEN: phospho-amino acid interactions (PerkinElmer) according to manufacturer’s instruction. Saturation curve and competition curve were generated to determine K_d of the interaction between biotin-PIP₃, biotin-BSA-pTyr, -pSer, -pThr or biotin-BSA as indicated and His6-PTEN in Alpha format, in which unlabeled PIP₃ (10 µM to 0.01 nM) and BSA-pTyr, -pSer, or -pThr (400 µM to 0.04 nM) were used as competitor, respectively. The saturation binding curve and competitive inhibition curve were calculated based on Alpha signal readings by fitting to a nonlinear regression “saturation binding” model and a “log (inhibitor) vs. response-Variable slope (four parameters)” model, respectively. The K_d values were calculated using GraphPad Prism 6 software.