Mogridge Jeremy
Department of Laboratory Medicine and Pathobiology, University of Toronto, Medical Sciences Building, Room 6308, 1 King's College Circle, Toronto, ON, Canada, M5S 1A8,
Methods Mol Biol. 2015;1278:233-8. doi: 10.1007/978-1-4939-2425-7_14.
The stoichiometry of a protein complex can be calculated from an accurate measurement of the complex's molecular weight. Multiangle laser light scattering in combination with size exclusion chromatography and interferometric refractometry provides a powerful means for determining the molecular weights of proteins and protein complexes. In contrast to conventional size exclusion chromatography and analytical centrifugation, measurements do not rely on the use of molecular weight standards and are not affected by the shape of the proteins. The technique is based on the direct relationship between the amount of light scattered by a protein in solution, and the product of its concentration and molecular weight. A typical experimental configuration includes a size exclusion column to fractionate the sample, a light scattering detector to measure scattered light, and an interferometric refractometer to measure protein concentration. The determination of the molecular weight of an anthrax toxin complex will be used to illustrate how multiangle laser light scattering can be used to determine the stoichiometry of protein complexes.
蛋白质复合物的化学计量比可通过对该复合物分子量的精确测量来计算。多角度激光光散射结合尺寸排阻色谱法和干涉折射法为测定蛋白质和蛋白质复合物的分子量提供了一种强大的手段。与传统的尺寸排阻色谱法和分析离心法不同，该测量方法不依赖于分子量标准品的使用，也不受蛋白质形状的影响。该技术基于溶液中蛋白质散射光的量与其浓度和分子量乘积之间的直接关系。典型的实验配置包括一个用于分离样品的尺寸排阻柱、一个用于测量散射光的光散射检测器和一个用于测量蛋白质浓度的干涉折射仪。炭疽毒素复合物分子量的测定将用于说明如何使用多角度激光光散射来确定蛋白质复合物的化学计量比。
