A novel experiment using affinity chromatography to separate a target protein from an unknown protein was developed for freshman in a foundation lab focusing on chromatographic separation techniques. In contrast to published experiments involving purification of a target protein from a complex mixture using an affinity column with a single bait ligand, students are given a binary mixture of a one of 10 different unknown proteins, one of 4 target proteins, and an affinity gel with a bait ligand for the assigned target. Targets include carbonic anhydrase, conalbumin, biotinylated lacatalbumin, and glutamate dehydrogenase, which bind specifically to 4-(aminomethyl)benzenesulfonamide-, 3-aminophenylboronic acid-, and avidin-conjugated Sepharose 4B-Cl and conjugated Cibachron Blue F3G-A. After elution, students analyze proteins in the initial mixture, fall-through, and final elution, and identify both target and unknown using SDS-polyacrylamide gel electrophoresis. Details of the experiment and its context within the new Foundation Purification 2 Laboratory will be addressed.

Purification and characterization of proteins by affinity chromatography in a first year laboratory

243rd National Meeting (2012)

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