Urea Gradient/SDS-Page; A Useful Tool in the Investigation of Signal Transducing G Proteins

Abstract

We describe an updated and improved protocol to perform urea gradient/SDS-PAGE in which proteins are electrophoresed through 9% polyacrylamide gel slabs in the presence of a linear 4 M to 8 M gradient of urea using Laemmli's separation buffers. We provide examples of this technique to separate PTX labeled G protein α subunits, as well as unlabeled α and β subunits of G proteins. Applications of the technique are exemplified in which (1) the chromatographic separations of G proteins in DEAE-Toyopearl and MonoQ columns are compared, (2) the complexity of PTX substrates expressed in human erythrocytes, bovine brain, dog ventricle, FRTL-5 cells, HIT cells, GH₄C₁ cells and RIN cells are compared, and (3) the polypeptide composition of G protein β_γ subunits, as expressed in several tissues and found in three distinct G proteins from a single cell population, are analyzed.