Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes.
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.
Swamy
Mahima
M
Max Planck-Institut für Immunbiologie und Universität Freiburg, Biologie III, Stübeweg 51, D-79108 Freiburg, Germany.
Siegers
Gabrielle M
GM
Minguet
Susana
S
Wollscheid
Bernd
B
Schamel
Wolfgang W A
WW
D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007202:Indicators and Reagents; D008565:Membrane Proteins / Q000302:isolation & purification; D046912:Multiprotein Complexes / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D019000:Phosphotyrosine / Q000276:immunology; D021122:Protein Subunits / Q000032:analysis; D011947:Receptors, Antigen, B-Cell / Q000032:analysis; D012394:Rosaniline Dyes; D016622:Silver Staining
D007202:Indicators and Reagents; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D011947:Receptors, Antigen, B-Cell; D012394:Rosaniline Dyes; D019000:Phosphotyrosine; C048139:Coomassie blue