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, and copper(II) sulfate pentahydrate.
The BCA assay primarily relies on two reactions. First, the peptide bonds in protein reduce Cu ions from the copper(II) sulfate to Cu (a temperature dependent reaction). The amount of Cu 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.
The amount of protein present in a solution can be quantified by measuring the absorption spectra and comparing with protein solutions of known concentration.