Autoradiogram showing results of phosphoamino acid analyses on the 12-kD erythrocyte membrane protein. Sephacryl S-300 column fractions no. 40 through 51 were concentrated 6-fold and allowed to undergo phosphorylation in the presence of protein kinase buffer containing 1 μmol/L [γ-³²P]ATP (1 × 10⁷dpm), 4 mmol/L magnesium acetate, and 1 mmol/L manganese acetate, without (−) or with (+) 1 μmol/L IP₃. Reaction mixtures were subsequently applied to 3% to 17% gradient SDS polyacrylamide gels and a 12-kD phosphoprotein was identified on autoradiograms. Gel segments containing this component were cut out and the phosphoproteins were hydrolyzed in 6 N HCl at 110°C for 65 minutes. Liberated phosphoamino acids were separated by thin-layer chromatography using the system absolute ethanol:25% ammonia (3.5:1.6).46 Authentic standards (phosphoserine, phosphothreonine, and phosphotyrosine) were cospotted in each sample lane and subsequently visualized with ninhydrin.