Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for the Identification and Analysis of Multiprotein Complexes

• DOI: 10.1126/stke.3452006pl4
• Publication Date: 2006-09-24T00:12:11Z
• Authors: Mahima Swamy; Gabrielle M. Siegers; Susana Minguet; Bernd Wollscheid; Wolfgang W. A. Sc...
• Keywords: 570; Silver Staining; 0303 health sciences; Blotting, Western; 610; Membrane Proteins; Receptors, Antigen, B-Cell; Protein Subunits; 03 medical and health sciences; /dk/atira/pure/subjectarea/asjc/2700/2700; Multiprotein Complexes; Rosaniline Dyes; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Indicators and Reagents; name=General Medicine; Phosphotyrosine

Multiprotein complexes (MPCs) play crucial roles in cell signaling. Two kinds of MPCs can be distinguished: (i) Constitutive, abundant MPCs--for example, multisubunit receptors or transcription factors; and (ii) signal-induced, transient, low copy number MPCs--for example, complexes that form upon binding of Src-homology 2 (SH2) domain-containing proteins to tyrosine-phosphorylated proteins. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a separation method with a higher resolution than gel filtration or sucrose density ultracentrifugation that can be used to analyze abundant, stable MPCs from 10 kD to 10 MD. In contrast to immunoprecipitation and two-hybrid approaches, it allows the determination of the size, the relative abundance, and the subunit composition of an MPC. In addition, it shows how many different complexes exist that share a common subunit, whether free monomeric forms of individual subunits exist, and whether these parameters change upon cell stimulation. Here, we give a detailed protocol for the separation of MPCs from total cellular lysates or of prepurified MPCs by one-dimensional BN-PAGE or by two-dimensional BN-PAGE and SDS-PAGE.