Spectrophotometric and Colorimetric Determination of Protein Concentration

Abstract

AbstractSpectrophotometric and Colorimetric Determination of Protein Concentration (Michael H. Simonian, Beckman‐Coulter, Inc., Fullerton, California, and John A. Smith, University of Alabama at Birmingham, Birmingham, Alabama). Measuring absorbance at 280 nm (A280) is one of the oldest methods for determining protein concentration. This method is still widely used because it is simple and does not require incubating the sample with exogenous chromophores. Accordingly, the quantitation of proteins by peptide bond absorption at 205 nm (A205) is more universally applicable. Furthermore, the absorptivity for a given protein at 205 nm is several‐fold greater than that at 280 nm. Thus, lower concentrations of protein can be quantitated with the A205 method. The disadvantage of this method is that some buffers and other components absorb at 205 nm. The aromatic amino acids also exhibit fluorescence emissions when excited by light in the UV range. Measurement of intrinsic fluorescence by aromatic amino acids is primarily used to obtain qualitative information. However, with a protein standard whose aromatic amino acid content is similar to that of the sample, intrinsic fluorescence can be used for quantitation. The most frequently employed colorimetric methods for determining protein concentration are those of Bradford and Lowry.