Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes.

Mahima Swamy, Gabrielle M. Siegers, Susana Minguet, Bernd Wollscheid, Wolfgang W.A. Schamel (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

100 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)pl4
JournalScience's STKE : signal transduction knowledge environment
Volume2006
Issue number345
DOIs
Publication statusPublished - 25 Jul 2006

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