Figure 1.

PI 3-kinase was affinity-purified from platelet cytosol. A peptide (Y751) corresponding to the p85-binding site on the platelet-derived growth factor receptor was coupled to Actigel Superflow resin. Six hundred microliters of human platelet cytosol (12 mg/ml) was incubated with 60 μl of a 50% slurry of the peptide-coupled resin. The resin was pelleted and washed with either (1) low-stringency or (2) high-stringency buffer. PI 3-kinase assays were performed on washed pellets in duplicate by using PtdIns 4-P as substrate.