Preparation of an Affinity Chromatographic Matrix

Affinity media with immobilized carbohydrates have been widely used for the separation of lectins in the final purification step in the multistep protocols. These affinity matrixes themselves are fairly expensive and contribute to the high purification costs. The present work shows a simple and inexpensive method for the preparation of an affinity chromatographic matrix for maltose-specific lectins. Agarose beads were prepared by emulsification technique where 4% w/v agarose was dissolved in boiling water containing 0.9% NaCl and used as water phase. A mixture of liquid paraffin containing 3 wt% of tween 80 emulsifier was used as oil phase. Beads with diameter from 40 to 80 ¿m were obtained in this method. Agarose gel beads were activated using epichlorohydrin as activation reagent, after which maltose was immobilized as an affinity ligand on the epoxy groups. The prepared affinity matrix was evaluated by purifying Canavalia gladiata lectin (CGL) after packed into a column. The extent of purification was judged by SDS-PAGE.