Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis.
The nucleus is a highly structured organelle with distinct compartmentalization of specific functions. To understand the functions of these nuclear compartments, detailed description of protein complexes which form these structures is of crucial importance. We explored therefore the potential of blue native PAGE (BN-PAGE) method for the separation of nuclear protein complexes. We focused on (i) solubility and stability of nuclear complexes under conditions prerequisite for the separation by BN-PAGE, (ii) improved separation of native nuclear protein complexes using 2-D colorless native/blue native PAGE (CN-/BN-PAGE), and (iii) mass spectrometric analysis of protein complexes which were isolated directly from native 1-D or from 2-D CN/BN-PAGE gels. The suitability of BN-PAGE for nuclear proteomic research is demonstrated by the successful separation of polymerase I and polymerase II complexes, and by mass spectrometric determination of U1 small nuclear ribonucleoprotein particle composition. Moreover, practical advice for sample preparation is provided.
Nováková
Zora
Z
Department of Cell Ultrastructure and Molecular Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Praque, Czech Republic.
Man
Petr
P
Novák
Petr
P
Hozák
Pavel
P
Hodný
Zdenek
Z
D000595:Amino Acid Sequence; D000818:Animals; D002021:Buffers; D002467:Cell Nucleus / Q000737:chemistry*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry; D009687:Nuclear Proteins / Q000302:isolation & purification*; D051381:Rats; D012995:Solubility
D002021:Buffers; D046912:Multiprotein Complexes; D009687:Nuclear Proteins