Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis

Electrophoresis
Zora NovákováZdenek Hodný

Abstract

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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Citations

Sep 29, 2011·Nature Methods·Svitlana MelnikPeter R Cook
Dec 17, 2008·Mass Spectrometry Reviews·Thierry RabilloudMireille Chevallet
Sep 4, 2008·Proteomics·Ilka Wittig, Hermann Schägger
Jul 24, 2012·Proteomics. Clinical Applications·Rui HuangXiao-Ming Yang
Oct 17, 2009·Proteomics·Ingrid MillerElisabetta Gianazza
Sep 28, 2007·Biochemical and Biophysical Research Communications·Brian S HookerChiann-Tso Lin
Jul 27, 2007·Proteomics·Jesús V JorrínMa Angeles Castillejo
Sep 26, 2007·Proteomics·Veronika Reisinger, Lutz Andreas Eichacker
Feb 26, 2016·Nature Protocols·Svitlana MelnikArgyris Papantonis
Feb 6, 2017·Current Protocols in Protein Science·Xiaoting LuoLiang-Jun Yan

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