SEPARATION OF NUCLEAR PROTEIN COMPLEXES BY BLUE NATIVE POLY-ACRYLAMIDE GEL ELECTROPHORESIS
Nováková Z.1, Hodný Z.1, Man P.2 and Hozák P.1
1Department of Cell Ultrastructure and Molecular Biology, Institute of Experimental Medicine, and 2Mass Spectrometry Laboratory, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic The nucleus of eukaryotic cell is functionally and structurally compartmentalized. To understand the structure and function of individual nuclear domains, the knowledge about the protein composition of complexes building up these structures is needed. We explored therefore the potential of previously developed method for separation of membrane protein complexes in native state, the blue native poly-acrylamide gel electrophoresis (BN-PAGE), for separation of large nuclear protein complexes coupled with mass spectrometry analysis. In this study we demonstrate that modifications in solubilization of nuclear complexes are prerequisite for efficient separation by BN-PAGE. A solution for another problem, the high protein background disabling the subsequent mass spectrometric identification of protein complex composition (due to uncomplete separation of very basic nuclear proteins such as histones), was also sought. The usefulness of the method in nuclear research is demonstrated by 1), immunological detection of RNA polymerase I and II complexes and other nuclear protein complexes separated by one-dimensional blue native electrophoresis, and 2), mass spectrometric identification of subunit composition of U1snRNP particle after two-dimensional combination of colorless and blue native PAGE.
