Protein Separation and Characterization Procedures

This chapter covers the principles, procedures, and applications of a variety of techniques used to separate, isolate, and characterize proteins. Separation and isolation techniques rely on differences in physical, biochemical, and structural properties including bulk density, solubility, size, charge, thermal stability, and adsorption characteristics of the protein. Commonly used protein separation and isolation techniques include the following: dry fractionation, differential solubility based on physicochemical properties (e.g., charge, surface properties, and thermal stability), ion-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, size-exclusion chromatography, membrane filtration, and electrophoresis. Proteins can be characterized by their amino acid composition and sequence, nutritional quality, structural characteristics, and functional properties. Chromatographic techniques are used to determine the amino acid composition of a protein. The nutritional quality of a protein is evaluated by its amino acid composition and protein digestibility following in vivo and in vitro assays and the determination of protein digestibility-corrected amino acid score (PDCAAS) or protein efficiency ratio (PER). Common tests to evaluate the structural properties of the protein include molecular weight distribution, denaturation, and surface properties. Finally, evaluation of protein functionality includes the assessment of the protein water or oil binding capacity, solubility and dispersibility, emulsification and foaming properties, and protein network formation.