Binding studies using filtration methods

Other filter binding assays, like the nitrocellulose binding assay, have been used very successfully to investigate the interaction of proteins with various nucleic acids (Riggs et al. 1970). This method does not rely on differences in size to separate free and bound species, but on the fact that proteins are selectively bound to the mem- 

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Because membrane filtration is the only currently acceptable method of sterilizing protein pharmaceuticals, the adsorption and inactivation of proteins on membranes is of particular concern during formulation development. Pitt [56] examined nonspecific protein binding of polymeric microporous membranes typically used in sterilization by membrane filtration. Nitrocellulose and nylon membranes had extremely high protein adsorption, followed by polysulfone, cellulose diacetate, and hydrophilic polyvinylidene fluoride membranes. In a subsequent study by Truskey et al. [46], protein conformational changes after filtration were observed by CD spectroscopy, particularly with nylon and polysulfone membrane filters. The conformational changes were related to the tendency of the membrane to adsorb the protein, although the precise mechanism was unclear. 

Fractionation Methods. Ultrafiltration and gel filtration are nondestructive methods which, based on limited experience, can be used for fractionation of mineral complexes from digests. In earlier studies mineral absorption on the gel material was a problem. Lonnerdal (30) introduced a method of treating dextran gels with sodium borohydride in order to eliminate the mineral-binding sites on the gel. In preliminary studies we have recovered more than 90 of Ca, Mg, Fe, Zn and P in samples applied to a borohydride-treated gel column (Sephadex G-50, Pharmacia Fine Chemicals, Piscataway, NJ). Recovery of Ca (Table IV) and Mg, Fe and Zn from ultrafiltration was also good. 

In this work, we have used the chromatographic method of Hummel and Dreyer (5) and modified it in order to study the simultaneous binding of ADP and ATP on coupling factor CFl. In the original method, a known quantity q of macromolecule is injected on a gel filtration column which is preequilibrated with a fixed concentration (A) of ligand. 

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