Bicinchoninic acid assay

The bicinchoninic acid assay (BCA assay), also known as the Smith assay, after its inventor, Paul K. Smith at the Pierce Chemical Company,^[1] is a biochemical assay for determining the total concentration of protein in a solution (0.5 μg/mL to 1.5 mg/mL), 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
• Copper(II) sulfate pentahydrate

The BCA assay primarily relies on two reactions.

First, the peptide bonds in protein reduce Cu²⁺ ions from the copper(II) sulfate to Cu⁺ (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 Cu⁺ ion, forming a purple-colored complex that strongly absorbs light at a wavelength of 562 nm.

The bicinchoninic acid Cu⁺ complex is influenced in protein samples by the presence of cysteine/cystine, tyrosine, and tryptophan side chains. At higher temperatures (37 to 60 °C), peptide bonds assist in the formation of the reaction complex. 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.^[2]

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

See also

• Biuret test

References

1. Smith, P.K.; et al. (1985). "Measurement of protein using bicinchoninic acid". Anal. Biochem. 150 (1): 76–85. doi:10.1016/0003-2697(85)90442-7. PMID 3843705.
2. Olsen BJ and Markwell J. (2007). "Assays for the Determination of Protein Concentration" (PDF). Current Protocols in Protein Science: 14–17.

• 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 (1): 231–7. doi:10.1016/0003-2697(88)90383-1. PMID 3245570.
• Stoscheck, CM. (1990). "Quantitation of Protein". Methods in Enzymology 182: 50–69. doi:10.1016/0076-6879(90)82008-P. PMID 2314256.

External links

• OpenWetWare
• BCA assay chemistry