用于膜蛋白的高分辨率天然聚丙烯酰胺凝胶电泳，可进行荧光检测和流体力学状态评估。

High-resolution Native-PAGE for membrane proteins capable of fluorescence detection and hydrodynamic state evaluation.

机构信息

Molecular Signaling Research Team, Structural Physiology Research Group, RIKEN SPring-8 Center, Kouto, Sayo, Hyogo 679-5148, Japan.

出版信息

Anal Biochem. 2011 May 15;412(2):217-23. doi: 10.1016/j.ab.2011.01.038. Epub 2011 Feb 1.

DOI:10.1016/j.ab.2011.01.038

PMID:21291856

Abstract

An improved native polyacrylamide gel electrophoresis (PAGE) method capable of evaluating the hydrodynamic states of membrane proteins and allowing in-gel fluorescence detection was established. In this method, bis(alkyl) sulfosuccinate is used to provide negative charges for detergent-solubilized membrane proteins to facilitate proper electrophoretic migration without disturbing their native hydrodynamic states. The method achieved high-resolution electrophoretic separation, in good agreement with the elution profiles obtained by size exclusion chromatography. The applicability of in-gel fluorescence detection for tagged green fluorescent protein (GFP) facilitates the analysis of samples without any purification. This method might serve as a general analytical technique for assessing the folding, oligomerization, and protein complex formation of membrane proteins.

摘要

建立了一种改良的原生聚丙烯酰胺凝胶电泳（PAGE）方法，能够评估膜蛋白的流体动力学状态，并允许胶内荧光检测。在该方法中，双（烷基）磺基琥珀酸盐为去污剂溶解的膜蛋白提供负电荷，以促进适当的电泳迁移，而不会干扰其天然的流体动力学状态。该方法实现了高分辨率电泳分离，与尺寸排阻色谱法获得的洗脱图谱非常吻合。胶内荧光检测标记的绿色荧光蛋白（GFP）的适用性便于分析未经任何纯化的样品。该方法可作为评估膜蛋白折叠、寡聚化和蛋白质复合物形成的通用分析技术。