U-2012: An improved Lowry protein assay, insensitive to sample color, offering reagent stability and enhanced sensitivity

Biotechniques. 2012 Mar;52(3):159-66. doi: 10.2144/000113818.

Abstract

Traditional colorimetric protein assays such as Biuret, Lowry, and modified Lowry (U-1988) are unsuitable for colored biological samples. Here we describe an improved Lowry protein assay (U-2012), which utilizes stable reagents and offers enhanced sensitivity over the U-1988 assay. U-2012 circumvents interference from colored pigments and other substances (for example sugars) bound to perchloric acid (PCA) precipitated proteins by hydrogen peroxide (H2O2) induced oxidation at 50°C. Unused hydrogen peroxide is neutralized with sodium pyruvate before protein estimation for a stable end color. The U-2012 assay is carried out on the PCA precipitated protein pellet after neutralization (with Na2CO3 plus NaOH), solubilization (in Triton-NaCl), decolorization (by H2O2) and pyruvate treatment. Protein contents in red wine and homogenates of beetroot and blueberry are calculated from standard curves established for various proteins and generated using a rectangular hyperbola with parameters estimated with Microsoft Excel's Solver add-in. The U-2012 protein assay represents an improvement over U-1988 and gives a more accurate estimation of protein content.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Beta vulgaris
  • Blueberry Plants
  • Chemical Precipitation
  • Chemistry Techniques, Analytical / methods*
  • Perchlorates / chemistry
  • Pigments, Biological
  • Plant Proteins / analysis*
  • Sensitivity and Specificity
  • Wine

Substances

  • Perchlorates
  • Pigments, Biological
  • Plant Proteins