Methods for Protein Quantitation

Abstract

Numerous methods for determining the amount of protein present in a sample have been devised over the years. Gravimetric methods for lyophilized (freeze dried) proteins have been used, as well as methods based on elemental analysis, such as the Kjeldahl nitrogen assay (Kjel-dahl, 1983). These methods have largely been replaced by colorimetric and spectroscopic methods which offer convenient and reasonably accurate estimations of protein content in solution. Since these are the most commonly used methods for protein quantitation, we shall discuss them in detail here. The reader who wishes to pursue other methods for protein concentration determinations is referred to the excellent review by Darbre (1986). A comparison of the most common methods for protein concentration analysis has recently been made by Bio-Rad Laboratories, and is summarized in table 3.1. COLORIMETRIC METHODS Colorimetric assays for proteins are based on the fact that certain metal ions and dyes bind to proteins in a specific mass ratio, and upon binding become intensely colored. Within a specific range of protein concentration , these reagents will give rise to an absorption band whose intensity is linearly proportional to the protein concentration of a solution in mass/ volume units. The three most commonly used of these methods are: the Biuret assay, the Lowry assay, and the Bradford assay. 39 R. A. Copeland, Methods for Protein Analysis