Korepanova Alla, Matayoshi Edmund D
Department of Biochemistry, Advanced Technologies, Abbott Laboratories, Abbott Park, Illinois.
Department of Structural Biology, Advanced Technologies, Abbott Laboratories, Abbott Park, Illinois.
Curr Protoc Protein Sci. 2012 Apr;Chapter 29:29.5.1-29.5.12. doi: 10.1002/0471140864.ps2905s68.
Determination of the oligomeric state of integral membrane proteins in detergent solutions is a challenging task because the amount of detergent associated with the protein is typically unknown and unpredictable. Methods that estimate the molecular weight of proteins from their hydrodynamic properties in solution are not suitable for detergent-solubilized membrane proteins. However, size-exclusion chromatography (SEC) performed in combination with analyses of static light scattering (SLS), ultraviolet absorbance (UV), and refractive index (RI) provides a universal method for determination of the molar masses of biopolymers and protein-detergent complexes. The light scattered by a protein is directly proportional to its molecular mass, irrespective of shape, and any additional contributions due to bound detergent molecules can be quantitatively accounted for by the additional combined analysis of ultraviolet absorbance and refractive index information. The primary intention of this unit is to describe how to apply the combination of high-performance liquid chromatography SEC and SLS-UV-RI to evaluate molecular mass and the physicochemical heterogeneity of purified membrane protein-detergent complexes.
确定去污剂溶液中整合膜蛋白的寡聚状态是一项具有挑战性的任务，因为与蛋白质结合的去污剂的量通常是未知且不可预测的。根据蛋白质在溶液中的流体力学性质估算其分子量的方法不适用于去污剂增溶的膜蛋白。然而，尺寸排阻色谱（SEC）结合静态光散射（SLS）、紫外吸收（UV）和折射率（RI）分析，为测定生物聚合物和蛋白质-去污剂复合物的摩尔质量提供了一种通用方法。蛋白质散射的光与其分子量成正比，与形状无关，并且由于结合的去污剂分子产生的任何额外贡献都可以通过紫外吸收和折射率信息的额外联合分析进行定量计算。本单元的主要目的是描述如何应用高效液相色谱SEC与SLS-UV-RI的组合来评估纯化的膜蛋白-去污剂复合物的分子量和物理化学异质性。
