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-s), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional coefficient) as the biomolecule. Determination of R-s by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca2+) to many Ca2+-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca2+ 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-s of proteins with some indications that are specific for Ca2+ sensor proteins.

Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography

La Verde, Valentina;Dominici, Paola;Astegno, Alessandra
2017-01-01

Abstract

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-s), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional coefficient) as the biomolecule. Determination of R-s by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca2+) to many Ca2+-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca2+ 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-s of proteins with some indications that are specific for Ca2+ sensor proteins.
2017
Size exclusion chromatography; Gel filtration; Hydrodynamics; Stokes radius; Protein shape; Protein size; Conformational change; Ca2+-sensor proteins
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/1011479
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