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:
* 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 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 (37^oC to 60^oC), 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

cite journal | author=Olsen BJ and Markwell J. | title= [http://media.wiley.com/CurrentProtocols/0471111848/0471111848-sampleUnit.pdf Assays for the Determination of Protein Concentration] | journal=Current Protocols in Protein Science | year=2007 | supplement=48 | pages=14–17
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