Well defined dye adsorbents for protein purification

When activating a matrix with epichlorohydrin, the degree of activation can be controlled by the amount of epichlorohydrin added and the length of time of the reaction. The epoxy group can react with thiols and amines to produce N- and S-linked adsorbents for protein purification. The epoxy-activated gel may also react with ammonia to produce an amino-activated adsorbent. This adsorbent reacts readily with the reactive groups of textile dyes, coupling the dyes to the matrix via a short spacer arm. Conditions can be found where the reaction of the dyes with hydroxyl groups on the matrix is minimized, producing dye-ligand adsorbents with well defined ligand densities. These dye adsorbents can be made with much higher ligand densities than are normally achievable with the conventional coupling via hydroxyl groups on the matrix. The protein-binding behaviour of the highly substituted adsorbents is qualitatively similar to that of the conventional adsorbents but the capacity increases with increase in ligand density. Too high a ligand density is undesirable as it may become difficult to elute the proteins from the adsorbent. The optimum ligand density was found to be between 2 and 10 mumol dye ligand per mL adsorbent, the levels chosen for the low- and high-dye kits of dye adsorbents for protein purification (Rainbow-sorb).