In this assay, proteins in the sample cause cupric ions (Cu²⁺) to be reduced to cuprous ions (Cu⁺); this is due to the electron-donating properties of peptide bonds and certain amino acids like cysteine, tyrosine, and tryptophan. This reduction occurs under alkaline conditions, where BCA then reacts with the cuprous ions to form a deep purple-colored complex. The intensity of this purple color is measured using a spectrophotometer at a wavelength of 562 nm. The color intensity is correlated directly with the concentration of protein in the sample, allowing for the quantification of protein levels within a range of 20–2000 µg/mL.