Springer Protocols: Abstract: Isolation of Membrane Protein Complexes by Blue Native Electrophoresis

Isolation of Membrane Protein Complexes by Blue Native Electrophoresis

By: Veronika Reisinger³, Lutz A. Eichacker³

Abstract

Full Text

Download PDF (359K)

Blue native PAGE is a discontinuous electrophoretic system that allows the separation of membrane protein complexes in a native, enzymatically active state with high resolution. Membrane protein complexes are solubilized by neutral, nondenaturing detergents like n-dodecyl- $\beta$ -d-maltoside. After addition of Coomassie G250 that binds to the surface of the proteins, separation of the negatively charged complexes according to molecular mass is possible. After electrophoresis the structure and function of the isolated protein complexes can be investigated.

Affiliation(s): (3) Ludwig-Maximalians-University Munich, Munich, Germany

Book Title: 2D PAGE: Sample Preparation and Fractionation

Series: Methods in Molecular Biology | Volume: 424 | Pub. Date: Jan-25-2008 | Page Range: 423-431 | DOI: 10.1007/978-1-60327-064-9_33

Subject: Genetics/Genomics

Key Words: BN PAGE - Coomassie G250 - electrophoresis - functional complex - membrane protein - n-dodecyl- $\beta$ -d-maltoside.

References

Download References

1.	Righetti, P. G. (2005) Electrophoresis: the march of pennies, the march of dimes. J. Chromatogr. A. 1079, 24–40.

2.	Zahedi, R. P., Meisinger, C. and Sickmann, A. (2005) Two-dimensional benzyldimethyl-n-hexadecylammonium chloride/SDS-PAGE for membrane proteomics. Proteomics, 5, 3581–3588.

3.	Schägger, H. and von Jagow, G. (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 166, 223–231.

4.	Schägger, H., Cramer, W. A. and von Jagow, G. (1994) Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane complexes by two dimensional native electrophoresis. Anal. Biochem. 217, 220–230.

5.	Brouillard, F., Bensalem, N., Hinzpeter, A., Tondelier, D., Trudel, S., Gruber, A. D., Ollero, M. and Edelman, A. (2005) Blue native-SDS PAGE analysis reveals reduced expression of the mClCA3 protein in cystic fibrosis knock-out mice. Mol Cell Proteomics. 4, 1762–1775.

6.	Eubel, H., Heinemeyer, J., Sunderhaus, S. and Braun, H. P. (2004) Respiratory chain supercomplexes in plant mitochondria. Plant Physiol Biochem. 42, 937–942.

7.	Komenda, J., Reisinger, V., Müller, B. C., Dobakova, M., Granvogl, B. and Eichacker, L. A. (2004) Accumulation of the D2 protein is a key regulatory step for assembly of the photosystem II reaction center complex in Synechocystis PCC 6803. J. Biol. Chem. 279, 48620–48629.

8.	Schägger, H. (2003) Blue Native Electrophoresis, in Membrane Protein Purification and Crystallization (Hunte, C., von Jagow, G. and Schägger, H., eds.), Elsevier Science, USA, pp. 105–130.

9.	Darie, C. C., Biniossek, M. L., Winter, V., Mutschler, B. and Haehnel, W. (2005) Isolation and structural characterization of the Ndh complex from mesophyll and bundle sheath chloroplasts of Zea mays. FEBS J. 272, 2705–2716.

10.	Jung, C., Higgins C.M. and Xu, Z. (2000) Measuring the quantity and activity of mitochondrial electron transport chain complexes in tissues of central nervous system using blue native polyacrylamide gel electrophoresis. Anal Biochem.. 286, 214–223.

Comments (Loading...)

Post comment/View all comments