Protein Concentration Determination Using Dyes and Beer-Lambert Law

Abstract

Synonyms Absorbance of light; Transmission of light

Definition Although proteins have different percentages of different amino acids, each residue has an amide bond linking it to the next residue in the chain. A number of concentration determination methods have been developed that involve derivatizing the amides with a dye and spectroscopically determining the concentration of the derivatives (Noble and Bailey 2009). The three methods mentioned below all rely on a standard of known concentration to enable a calibration curve to be plotted. The calibration is not necessarily linear and ideally is the same protein of known purity since the dye binding efficiency varies from protein to protein. Other reagents in the sample such as buffer salts and detergents may also interfere with either the chemistry or the spectroscopy of the dyes.

Basic Characteristics The methods for three commonly used assays are outlined below.

Biuret Method The biuret method is simple and reasonably specific as...