Bicinchoninic acid assay

First, the peptide bonds in protein reduce Cu2+ ions from the copper(II) sulfate to Cu1+ (a temperature dependent reaction).

Next, two molecules of bicinchoninic acid chelate with each Cu1+ ion, forming a purple-colored complex that strongly absorbs light at a wavelength of 562 nm.

The bicinchoninic acid Cu1+ complex is influenced in protein samples by the presence of cysteine/cystine, tyrosine, and tryptophan side chains.

The BCA assay is largely incompatible with reducing agents and metal chelators, although trace quantities may be tolerated.

Reagent A[1] A suggested but untested alternative formulation in the Smith manuscript is to leave out the NaOH (and presumably not perform the manual pH adjustment to 11.25), but instead to dissolve the other components in a preprepared buffer of 0.25 M Na2CO3 and 0.01 M NaHCO3.

[9][10] Notably, the composition and use of a "Micro BCA Reagent and Protocol" was described in the original manuscript by Smith,[1] and modern kits likely consist of an exact or highly similar formulation.

[4] The Pierce Quantitative Colorimetric Peptide Assay (now owned by and available from Thermo Fisher Scientific) appears to use a similar or identical 480 nm absorbing proprietary copper chelator.