Protein measurement using bicinchoninic acid: elimination of interfering substances

Rhoderick E Brown, Kari L. Jarvis, Kristi J. Hyland

Research output: Contribution to journalArticle

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Abstract

Protein quantitation based on bicinchoninic acid (BCA) is simple, sensitive, and tolerant to many detergents and substances known to interfere with the Lowry method. However, certain compounds often used during protein purification do interfere with the BCA protein assay. The response of the BCA chromophore to various interfering substances has provided insight into the mechanism of protein quantitation by BCA. Certain substances (e.g., glucose, mercaptoethanol, and dithiothreitol) elicit a strong absorbance at 562 nm when combined with the BCA working reagent. The absorbance appears to be identical to the normal response elicited by protein. Other agents (e.g., ammonium sulfate and certain ampholytes) diminish the protein-induced color development and shift the wavelength of the color response. Both types of interference can be eliminated by selectively precipitating protein with deoxycholate and trichloroacetic acid (A. Bensadoun and D. Weinstein (1976) Anal. Biochem. 70, 241-250) prior to reaction with bicinchoninic acid. The modifications described here permit quick, efficient removal of many interfering substances that are commonly utilized during protein purification.

Original languageEnglish (US)
Pages (from-to)136-139
Number of pages4
JournalAnalytical Biochemistry
Volume180
Issue number1
DOIs
StatePublished - Jul 1989

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