Separations gel filtration

GF is well suited for use after any of the concentrating techniques (IEX, HIC, AC, EBA) since the target protein will be eluted in a reduced volume and the components from the elution buffer will not affect the gel filtration separation (gel filtration is a non-binding technique with limited volume capacity and unaffected by buffer conditions). 

An alternative to dialysis methods is the gel filtration technique of Hummel and Dreyer (57), as modified by Price (58). A column of Sephadex G-25 is equilibrated with a buffer containing a desired concentration of calcium and 45CaCl2, and is used for gel filtration of the binding protein. As the process of gel filtration proceeds, the protein migrates in the excluded volume of the column, removing calcium ions from the column buffer until equilibrium is reached. The protein peak, in the void volume of the column, contains above-base line amounts of calcium. The amount of calcium bound to the protein can be found by dividing the molar concentration of calcium above the base line value by the molar protein concentration. A separate gel filtration run is used to determine each point on the binding plot. This is at once time-consum-... 

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