Okumoto Kanji, Tamura Shigehiko, Fujiki Yukio
Department of Biology, Faculty of Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.
Division for Experimental Natural Science, Faculty of Arts and Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.
Methods Mol Biol. 2017;1595:197-205. doi: 10.1007/978-1-4939-6937-1_18.
Blue native polyacrylamide gel electrophoresis (BN-PAGE) is one of the useful methods to isolate protein complexes including membrane proteins under native conditions. In BN-PAGE, Coomassie Brilliant Blue G-250 binds to proteins and provides a negative charge for the electrophoretic separation without denaturing at neutral pH, allowing the analysis of molecular mass, oligomeric state, and composition of native protein complexes. BN-PAGE is widely applied to the characterization of soluble protein complexes as well as isolation of membrane protein complexes from biological membranes such as the complexes I-V of the mitochondrial respiratory chain and subcomplexes of the mitochondrial protein import machinery. BN-PAGE has also been introduced in the field of peroxisome research, for example, analysis of translocation machinery for peroxisomal matrix proteins embedded in the peroxisomal membrane. Here, we describe a basic protocol of BN-PAGE and its application to the study of peroxisome biogenesis.
蓝色天然聚丙烯酰胺凝胶电泳（BN-PAGE）是在天然条件下分离包括膜蛋白在内的蛋白质复合物的有用方法之一。在BN-PAGE中，考马斯亮蓝G-250与蛋白质结合，并在中性pH下不为变性的情况下为电泳分离提供负电荷，从而能够分析天然蛋白质复合物的分子量、寡聚状态和组成。BN-PAGE广泛应用于可溶性蛋白质复合物的表征以及从生物膜中分离膜蛋白复合物，如线粒体呼吸链的复合物I-V和线粒体蛋白质导入机制的亚复合物。BN-PAGE也已引入过氧化物酶体研究领域，例如，分析嵌入过氧化物酶体膜中的过氧化物酶体基质蛋白的转运机制。在此，我们描述BN-PAGE的基本方案及其在过氧化物酶体生物发生研究中的应用。
