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Bicinchoninic_acid_assay

Bicinchoninic acid assay

The bicinchoninic acid assay (also known as the BCA assay or Smith assay) is a biochemical assay for determining the total level of protein in a solution, similar to Lowry protein assay, Bradford protein assay or biuret reagent. The total protein concentration is exhibited by a color change of the sample solution from green to purple in proportion to protein concentration, which can then be measured using colorimetric techniques.

Mechanism

A stock BCA solution contains the following ingredients in a highly alkaline solution with a pH 11.25:

The BCA assay primarily relies on two reactions.

Firstly, the peptide bonds in protein reduce Cu2+ ions from the cupric sulfate to Cu1+ (a temperature dependent reaction). The amount of Cu2+ reduced is proportional to the amount of protein present in the solution. Next, two molecules of bicinchoninic acid chelate with each Cu1+ ion, forming a purple-colored product that strongly absorbs light at a wavelength of 562 nm.

The bicinchoninic acid Cu1+ complex is aided in protein samples by the presence of cysteine, cystine, tyrosine, and tryptophan side chains. At higher temperatures (37oC to 60oC), peptide bonds assist in the formation of the reaction product. Incubating the BCA assay at higher temperatures is recommended as a way to increase assay sensitivity while minimizing the variances caused by unequal amino acid composition (Olsen and Markwell, 2007).

The amount of protein present in a solution can be quantified by measuring the absorption spectra and comparing with protein solutions with known concentrations.

References

Olsen BJ and Markwell J. (2007). "Assays for the Determination of Protein Concentration". Current Protocols in Protein Science 14–17.

  • Smith, P.K., et al. (1985). "Measurement of protein using bicinchoninic acid". Anal. Biochem. 150 76–85.
  • Wiechelman, K., Braun, R. and Fitzpatrick, J. (1988). "Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation". Anal. Biochem. 175 231–7.
  • Stoscheck, CM. (1990). "Quantitation of Protein". Methods in Enzymology 182 50–69.

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