Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis
Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis
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DOI：
10.1002/elps.200500504
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发表时间：
2006-04-01
期刊：
影响因子：
2.9
通讯作者：
Hodny, Z
中科院分区：
文献类型：
--
作者：
Nováková, Z;Man, P;Hodny, Z
细胞核是一个高度结构化的细胞器，具有不同的特定功能划分。为了了解这些核隔室的功能，详细描述形成这些结构的蛋白质复合体是至关重要的。因此，我们探索了蓝色天然PAGE(BN-PAGE)方法用于分离核蛋白复合体的潜力。我们重点研究了(I)核蛋白复合体在BN-PAGE分离条件下的溶解性和稳定性，(Ii)改进了利用二维无色天然/蓝色天然PAGE(CN-/BN-PAGE)分离天然核蛋白复合体的方法，以及(Iii)直接从天然一维或从天然CN/BN-PAGE凝胶中分离的蛋白质复合体的质谱分析。BN-PAGE对聚合酶I和聚合酶II复合体的成功分离，以及对U1小核核糖核蛋白颗粒组成的质谱分析，证明了BN-PAGE在核蛋白质组研究中的适用性。此外，还为样品的制备提供了实用的建议。
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.