Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
Subscribe

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

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).

Read the Full Text

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
The Biarylpyrazole Compound AM251 Alters Mitochondrial Physiology via Proteolytic Degradation of ERR{alpha}.
S. M. Krzysik-Walker, I. Gonzalez-Mariscal, M. Scheibye-Knudsen, F. E. Indig, and M. Bernier (2013)
Mol. Pharmacol. 83, 157-166
   Abstract »    Full Text »    PDF »
Cholesterol and Sphingomyelin Drive Ligand-independent T-cell Antigen Receptor Nanoclustering.
E. Molnar, M. Swamy, M. Holzer, K. Beck-Garcia, R. Worch, C. Thiele, G. Guigas, K. Boye, I. F. Luescher, P. Schwille, et al. (2012)
J. Biol. Chem. 287, 42664-42674
   Abstract »    Full Text »    PDF »
Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex.
R. Soundararajan, T. Ziera, E. Koo, K. Ling, J. Wang, S. A. Borden, and D. Pearce (2012)
J. Biol. Chem. 287, 33014-33025
   Abstract »    Full Text »    PDF »
Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter.
Z. Hou, S. Kugel Desmoulin, E. Etnyre, M. Olive, B. Hsiung, C. Cherian, P. A. Wloszczynski, K. Moin, and L. H. Matherly (2012)
J. Biol. Chem. 287, 4982-4995
   Abstract »    Full Text »    PDF »
hMSH2 Recruits ATR to DNA Damage Sites for Activation during DNA Damage-induced Apoptosis.
N. Pabla, Z. Ma, M. A. McIlhatton, R. Fishel, and Z. Dong (2011)
J. Biol. Chem. 286, 10411-10418
   Abstract »    Full Text »    PDF »
Oligomerization of Cav{beta} subunits is an essential correlate of Ca2+ channel activity.
Q. Z. Lao, E. Kobrinsky, Z. Liu, and N. M. Soldatov (2010)
FASEB J 24, 5013-5023
   Abstract »    Full Text »    PDF »
TIP47 functions in the biogenesis of lipid droplets.
A. V. Bulankina, A. Deggerich, D. Wenzel, K. Mutenda, J. G. Wittmann, M. G. Rudolph, K. N.J. Burger, and S. Honing (2009)
J. Cell Biol. 185, 641-655
   Abstract »    Full Text »    PDF »
Immunoreactivity of Phage Library-derived Human Single-Chain Antibodies to Amyloid Beta Conformers In Vitro.
T. Yoshihara, S. Takiguchi, A. Kyuno, K. Tanaka, S. Kuba, S. Hashiguchi, Y. Ito, T. Hashimoto, T. Iwatsubo, S. Tsuyama, et al. (2008)
J. Biochem. 143, 475-486
   Abstract »    Full Text »    PDF »
Different composition of the human and the mouse {gamma}{delta} T cell receptor explains different phenotypes of CD3{gamma} and CD3{delta} immunodeficiencies.
G. M. Siegers, M. Swamy, E. Fernandez-Malave, S. Minguet, S. Rathmann, A. C. Guardo, V. Perez-Flores, J. R. Regueiro, B. Alarcon, P. Fisch, et al. (2007)
J. Exp. Med. 204, 2537-2544
   Abstract »    Full Text »    PDF »
High-Sensitivity Detection and Quantitative Analysis of Native Protein-Protein Interactions and Multiprotein Complexes by Flow Cytometry.
A. G. Schrum, D. Gil, E. P. Dopfer, D. L. Wiest, L. A. Turka, W. W. A. Schamel, and E. Palmer (2007)
Sci. STKE 2007, pl2
   Abstract »    Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882