Bicinchoninic acid assay

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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.

[edit] 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.

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.

[edit] 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. doi:10.1016/0003-2697(85)90442-7. 
• 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. doi:10.1016/0003-2697(88)90383-1. 
• Stoscheck, CM. (1990). "Quantitation of Protein". Methods in Enzymology 182: 50–69.