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.

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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 dependant 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.

At the same time a second (temperature independant) reaction takes place between the reagent and certain side chains (including tyrosine, tryptophan). In order to minimise the effects of this sequence-specific reaction on the assay it is usually carried out at 37 degrees.

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

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.