Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis

Department of Cell Ultrastructure and Molecular Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Praque, Czech Republic.
Electrophoresis (Impact Factor: 3.03). 04/2006; 27(7):1277-87. DOI: 10.1002/elps.200500504
Source: PubMed


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.

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    • "In addition the size variability is also indicated by the presence of the PMH proteins in different fractions of the sucrose gradients, suggesting the size variation not to be the result of a particular preparative procedure. This distribution is highly reproducible and independent from the presence of aminocaproate which can destabilize protein complexes (data not shown) (Novakova et al., 2006). "
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