Purification of lysozyme by linear salt gradient and SDS gel electrophoresis

Abstract

I n order to provide the students in the introductory biochemistry lab with a sense of the approaches used to characterize and study biomolecules, we are adopting a project format focused on each class of biomolecules. The protein purification project is based upon the purification of hen egg white lysozyme. The purification of hen egg white lysozyme is routinely used to introduce the student to iou-exchange chromatography and enzyme purification.We have develoved a ~urification of lvsozvme that uti-. .lizes salt gradient elution ion-exchange chrorn:it(~@xphy nnd SDS n~l eiectro~horesis to sewrate und characterize the of hen egg white by &oionic point, molecular weight and by enzyme activity. Egg white lysozyme is purified on a carboxymethyl(CM)-Sephadex column eluted with a linear NaCl gradient. The resulting fractions are assayed for protein and lysozyme activity in order to identify the lysozyme active fractions. The lysozyme-active fractions are analyzed with SDS polyacrylamide gel electrophoresis for purity, and the molecular weights of the isolated proteins are determined.Reagents and Equipment SDS-polyacrylamide gel electrophoresis was run on an Ephortec electrophoresis apparatus from Buchler. Spectral readings for the Bradford protein assays and the lysozyme activity assays were done on a Bausch and LombPurification of Lysoyrne Fraction[protein] total activity specific (mg/mL)

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