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The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
442
Issue
1
Identifiers
DOI: 10.1016/j.ab.2013.07.027
Keywords
  • Urobilin
  • Bicinchoninic Acid
  • Protein Determination
  • Bile Pigment
  • Bradford
  • Limits Of Agreement Analysis
Disciplines
  • Biology
  • Design

Abstract

Abstract Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu2+) to cuprous ions (Cu1+), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.

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