Purification of Proteins Using UltraMacro Spin Columns or ProSorb Sample Preparation Cartridges for Amino Acid Analysis

Abstract

Amino acid analysis of proteins is one of the best and most accurate methodologies to quantify proteins. Ideally, samples should be pure prior to hydrolysis, however, they are often not only at low concentration, but also in buffers, salts, and/or detergents. Sample contaminants contribute to background noise that hampers amino acid analysis, variably affecting recoveries of individual amino acids (1). The necessity to obtain samples in high concentration and free of contaminants is a problem for protein quantitation and structural characterization (see Note 1). Many of the existing methods for removing buffers and salts from proteins, such as precipitation methods or reverse-phase high-performance liquid chromatography (RP-HPLC) result in variable and unacceptably high losses of protein (see Note 2), making an assessment of the true concentration difficult. In this chapter, we describe two methods commonly used to recover proteins from buffers: a gel filtration method, Ultra microspin columns, and adsorption to a polyvinylidene difluoride (PVDF) membrane in the ProSorb sample preparation cartridge from Applied Biosystems. Both methods work well, with somewhat better recoveries for the gel filtration method.