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A Practical Introduction to Structure, Mechanism, and Data Analysis - Part 4

Kích thước loại trừ sắc ký Kích thước loại trừ sắc ký thường được sử dụng với protein riêng biệt từ các loài trọng lượng phân tử nhỏ trong những gì được gọi là phương pháp protein-khử muối | 102 PROTEIN-LIGAND BINDING EQUILIBRIA 4.7.3 Size Exclusion Chromatography Size exclusion chromatography is commonly used to separate proteins from small molecular weight species in what are referred to as protein desalting methods see Copeland 1994 and Chapter 7 . Because of the nature of the stationary phase in these columns macromolecules are excluded and pass through the columns in the void volume. Small molecular weight species such as salts or free ligand molecules are retained longer within the stationary phase. Traditional size exclusion chromatography requires tens of minutes to hours to perform and is thus usually inappropriate for ligand binding measurements. Two variations of size exclusion chromatography are however quite useful for this purpose. In the first variation that is useful for ligand binding measurements spin columns are employed for size exclusion chromatography Penefsky 1977 Zeeberg and Caplow 1979 Andesson and Vaughan 1982 Copeland 1994 . Here a small bed volume size exclusion column is constructed within a column tube that fits conveniently into a microcentrifuge tube. Separation of excluded and retained materials is accomplished by centrifugal force rather than by gravity or peristaltic pressure as in conventional chromatography. After the column has been equilibrated with buffer a sample of the equilibrated receptor-ligand mixture is applied to the column. A separate sample of the mixture is retained for measurement of total ligand concentration. The column is then centrifuged according to the manufacturer s instructions and the excluded material is collected at the bottom of the microcentrifuge tube. This excluded material contains the protein-bound ligand population. By quantifying the ligand concentration in the sample before centrifugation and in the excluded material one can determine the total and bound ligand concentrations respectively. Again by subtraction one can also calculate the free ligand concentration and thus determine