La Verde Valentina, Dominici Paola, Astegno Alessandra
Department of Biotechnology, University of Verona, Verona, Italy.
Bio Protoc. 2017 Apr 20;7(8):e2230. doi: 10.21769/BioProtoc.2230.
Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (R), which is the radius of a sphere with the same hydrodynamic properties (, frictional coefficient) as the biomolecule. Determination of R by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca) to many Ca-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca binding, but likely arises from changes in the hydration shell structure. This protocol aims to describe a gel filtration experiment on a prepacked column using a Fast Protein Liquid Chromatography (FPLC) system to determine the R of proteins with some indications that are specific for Ca sensor proteins.
尺寸排阻色谱法（SEC）或凝胶过滤法是一种流体动力学技术，它根据分子的大小和形状在溶液中对分子进行分离。对于蛋白质而言，可通过实验得出的流体动力学值是斯托克斯半径（R），它是一个与生物分子具有相同流体动力学性质（即摩擦系数）的球体的半径。通过SEC测定R已被广泛用于监测钙（Ca）与许多钙传感蛋白结合所诱导的构象变化。对于这类蛋白质，SEC分离不仅基于钙结合后蛋白质大小的变化，还可能源于水化层结构的改变。本方案旨在描述使用快速蛋白质液相色谱（FPLC）系统在预装柱上进行的凝胶过滤实验，以确定蛋白质的R，并给出一些针对钙传感蛋白的特定指示。
