Streamline BCA-Based Protein Quantitation on the SpectraMax iD5 Reader

The BCA assay is a two-step colorimetric assay used to quantitate the total protein in a sample. This assay uses the Biuret reaction, in which Cu²⁺ is reduced to Cu¹⁺ by protein in an alkaline medium. The amino acid backbone forms a color-chelate complex with the copper molecules, allowing for a highly sensitive and selective detection of the cuprous cation. The resulting water-soluble complex exhibits strong absorbance which can be measured on a microplate reader. The original Pierce™ BCA Protein Assay Kit requires incubation for 30 minutes at 37°C and produces a color change that is measured at 562 nm. A newer version of the assay, the Pierce Rapid Gold BCA Protein Assay Kit, simply requires incubation for five minutes at room temperature and yields an intense orange-gold color that is measured at 480 nm.