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

doi:10.1126/stke.3452006pl4

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Sci. STKE, 25 July 2006
Vol. 2006, Issue 345, p. pl4
[DOI: 10.1126/stke.3452006pl4]

PROTOCOLS

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

Mahima Swamy¹, Gabrielle M. Siegers¹, Susana Minguet¹, Bernd Wollscheid², and Wolfgang W. A. Schamel¹^*

¹Max Planck-Institut für Immunbiologie und Universität Freiburg, Biologie III, Stübeweg 51, D-79108 Freiburg, Germany.
²NCCR Neuro Center for Proteomics, Institute for Molecular Systems Biology ETH Hönggerberg, Wolfgang Pauli-Str. 16, CH-8093 Zürich, Switzerland.

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.

*Corresponding author. Telephone, 49-761-5108-313; fax, 49-761-5108-423; e-mail, schamel{at}immunbio.mpg.de

Citation: M. Swamy, G. M. Siegers, S. Minguet, B. Wollscheid, W. W. A. Schamel, Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for the Identification and Analysis of Multiprotein Complexes. Sci. STKE 2006, pl4 (2006).

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