Isolation of phosphorylated peptides and proteins on ion exchange papers.

A simple technique for the isolation of ³²P-labeled peptides has been adapted from the ion exchange method of Witt and Roskoski (Anal. Biochem.66, 253, 1975). Protein kinase reaction mixtures are acidified and pipetted onto phosphocellulose papers. A variety of peptide and protein substrates are shown to bind to the ion exchange papers under conditions in which contaminating [γ-³²P]ATP is removed by washing in acetic acid. The capacity of phosphocellulose papers for the phosphopeptide, Leu-Arg-Arg-Ala-Ser(P)-Leu-Gly, exceeds 375 nmol per paper (2 × 2 cm). The method is limited to relatively basic peptides but is applicable to most proteins. Large numbers of samples can be processed simultaneously with no compromise in sensitivity or reliability of results.