Two-Dimensional Gel Electrophoresis for Analysis of Protein Complexes

Abstract

Advances in genomic and proteomic technologies combined with molecular and cell biology have together enabled the identification of numerous genes and their products. Two-dimensional gel electrophoresis (2DE) is especially useful in the study of protein-protein interactions as it permits an improved separation of proteins as well as the detection of specific interacting protein isoform(s) of a protein resulting from post-translational modification. The investigation of interacting proteins using 2DE can be complemented by identification of the proteins by mass spectrometry. Here, I describe how protein complexes, isolated by methods such as immunopre-cipitation, can be analyzed by 2DE using either isoelectric focusing (tube gels or immobilized pH gradient strips) or nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension, SDS-PAGE in the second dimension, and gel staining (silver and Coomassie) or Western blotting for the final detection of the interacting proteins.