The Bicinchoninic acid assay or BCA assay is a biochemical assay for detecting the presence of protein in a solution, similar to Lowry protein assay, Bradford protein assay or biuret reagent. It changes colour from green to purple in proportion to protein concentration in a given sample, which can then be measured using colorimetric techniques.

Procedure

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A stock BCA solution contains 1 g bicinchoninic acid (BCA), 2 g sodium carbonate, 0.16 g sodium tartrate, 0.4 g NaOH, and 0.95 g sodium bicarbonate per 100 ml of aqueous solution. The pH of the solution should be 11.25 (adjust with NaOH). The BCA solution is mixed in 50:1 ratio to 4% weight/volume CuSO4*5H2O solution to obtain a working solution.

In this preparation, 1 ml of the working solution and 20 μl of the protein solution to be assayed are mixed and incubated (2h at room temperature, 30 minutes at 37 °C, or 15 minutes at 60 °C) and then allowed to cool.

Kits are also available from numerous commercial sources. Typically they are more concentrated, and assays are performed in 200 μl volumes within a 96-well plate for higher thoroughput.

The BCA assay relies on two reactions. First, the Cu²⁺ ions, BCA and the peptide bonds or some of the amino acid residues in protein form a complex which reduces Cu²⁺ to Cu⁺¹. The amount of Cu²⁺ reduction is proportional to the amount of protein present in the solution. Then the Cu⁺¹ ion and BCA form a complex which has a purple-blue color and strong absorption at 562 nm. 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

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• P.K. Smith et al. Anal. Biochem. 150: 76 (1985)
• K. J. Wiechelman et al. Anal. Biochem. 175: 231 (1988)
• Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990)

biochemistry | chemical tests

Bicinchoninsäure