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 (32P-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. The methods have been developed using a small, basic phosphoprotein (histone H1 from Tetrahymena); however, the approach is probably applicable to a wide variety of phosphoproteins.
Dadd
C A
CA
Department of Biology, Syracuse University, NY 13244.
Cook
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RG
Allis
C D
CD
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017421:Sequence Analysis / Q000379:methods; D016808:Tetrahymena thermophila / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism*
D006657:Histones; D010750:Phosphoproteins; D014357:Trypsin