A Static Method as an Alternative to Gel Chromatography. An Experiment for the Undergraduate Biochemistry Laboratory

Abstract

This article describes a static method as an alternative to gel chromatography, which may be used as an undergraduate laboratory experiment. In this method, a constant mass of Sephadex gel is swollen in a series of protein solutions. UV–vis spectrophotometry is used to find a partition coefficient, KD, that indicates the fraction of the interior space of the gel bead available to the protein molecule. For a large protein that penetrates the bead pores only marginally, the partition coefficient will be small. For a small protein that penetrates substantially into the interior pore space, the partition coefficient will be large. In practice, a constant weighed quantity of dry gel is swollen in a series of protein solutions of different volumes. After equilibration, the supernatant is sampled and the absorbance at 280 nm is measured and compared to the absorbance of the original protein solution. A linear plot is made according to equations developed in this article, and the partition coefficient is found from the intercept. The method is reliable so long as sufficient care is taken in sample preparation.