The Identification of Phosphorylation Sites in Large Membrane Proteins Following Their Isolation by SDS-PAGE

Abstract

This chapter presents a study involving the identification of phosphorylation sites in large membrane proteins following their isolation by SDS-PAGE. Rat brain sodium channels were partially purified through the step of wheat germ agglutinin chromatography. A preparation containing 1200 pmol sodium channels was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase in the presence of 0.5 mM [γ-32P]ATP for 30 min at 37°C. The reaction was stopped by the addition of SDS-PAGE sample buffer and incubation at 55°C for 15 min. Rabbit skeletal muscle L-type calcium channels were purified, and a preparation containing 500 pmol was phosphorylated for the sodium channel. The primary structure of the rat brain type II sodium channel α subunit predicts six potential cAMP-dependent phosphorylation sites between residues 554 and 687.