BICINCHONINIC ACID ASSAY

The 'bicinchoninic acid assay' or 'BCA 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.

Contents

Mechanism

References

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 relies on two reactions. First, the peptide bonds in protein reduce Cu²⁺ ions from the cupric sulfate to Cu¹⁺. 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 amount of protein present in a solution can be quantified by measuring the absorption spectra and comparing with protein solutions with known concentrations.

References

★ Measurement of protein using bicinchoninic acid, Smith, P.K., ''et al''., , , Anal. Biochem., 1985

★ Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation, Wiechelman, K., Braun, R. and Fitzpatrick, J., , , Anal. Biochem., 1988

★ Quantitation of Protein, Stoscheck, CM., , , Methods in Enzymology, 1990