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:

• Bicinchoninic acid
• Sodium carbonate
• Sodium bicarbonate
• Sodium tartrate
• Cupric sulfate pentahydrate

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.