Fractionation of small tryptic phosphopeptides by alkaline PAGE followed by amino acid sequencing

A novel two-step approach for localizing the site(s) of phosphorylation within intact proteins is described. Phosphorylated ( 32 P-labeled) tryptic peptides are first resolved in a high-percentage polyacrylamide gel that has been optimized for the enrichment and separation of small, negatively charged peptides. Then the resolved peptides are located by autoradiography, excised, eluted and immobilized on a positively charged membrane, Immobilon®-N, where they can be sequenced directly