An improved Lowry protein assay, insensitive to sample color, offering reagent stability and enhanced sensitivity

www.BioTechniques.com 159 Vol. 52 | No. 3 | 2012 Quantitative measures of physiological traits such as enzyme activity are often expressed as units of activity per milligram protein. Although numerous assays have been developed to measure protein content, including the colorimetric assays of Amido Black (1), Biuret (2), Bicinchoninic Acid (3) and Coomassie Blue (4,5), the Lowry assay (6) or its modifications (7,8) are more commonly used than other assays (9). The Lowry assay is simple, sensitive and precise, and is the most cited (10) procedure for quantitative protein determination. A wide variety of compounds that react with Folin-Ciocalteu phenol (Folin’s) reagent (11) are a source of potential interference in Lowry and modified Lowry protein assays. Fortunately, corrections through an appropriate blank is sufficient for most compounds (6,7) except lipids (12), detergents (13) and colored substances (14). Difficulties in assaying proteins in presence of lipids and detergents (used in the solubilization of adipose tissue, myelin and skeletal muscles) were overcome by the modified Lowry assay (15; referred to in this paper as the U-1988 assay, 16). Color interference in determining the protein content in red wine (14,17,18) was overcome by employing extensive chromatography. The above approach is cumbersome and not very practical for handling large numbers of samples. None of the known protein assays were suitable for measuring proteins in colored biological samples e.g., colored fruits and vegetables, red wine, pigmented microbes and ruminant bile. Our development of the U-2012 assay from its predecessors the U-1988 and the Lowry assay has achieved three major advantages (i) convenience through stability of the reagent formulations, (ii) measurement of protein in both colorless and colored biological samples without compromising the sensitivity, and (iii) assaying proteins at very low concentrations. This novel assay will be applicable to quantitative determination of protein in both colorless and colored biological sample homogenates, including those rich in lipids (e.g., avocado) and those difficult to homogenize.