期刊
ANALYTICAL BIOCHEMISTRY
卷 442, 期 1, 页码 110-117出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2013.07.027
关键词
Urobilin; Bicinchoninic acid; Protein determination; Bile pigment; Bradford; Limits of agreement analysis
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. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved.
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