BCA Principle:

The Pierce BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. This method combines the well-known reduction of Cu⁺² to Cu⁺¹ by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu⁺¹) using a unique reagent containing bicinchoninic acid. The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562 nm that is nearly linear with increasing protein concentrations over a broad working range (20-2,000 μg/ml). The BCA method is not a true end-point method; that is, the final color continues to develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be assayed together. The macromolecular structure of protein, the number of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible for color formation with BCA. Studies with di-, tri- and tetrapeptides suggest that the extent of color formation caused by more than the mere sum of individual colorproducing functional groups.

Accordingly, protein concentrations generally are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA). A series of dilutions of known concentration are prepared from the protein and assayed alongside the unknown(s) before the concentration of each unknown is determined based on the standard curve.

BCA Assay Protocol:

Prepare working solution:

Mix reagent A (in general the blue bottle in a BCA kit) and reagent B with ratio of A:B=1:20 for enough volume of using.

Pipette working solution into microplate wells (100μl/well)

• • Add gradient volume of standard protein (2mg/ml) to each well (5 or more) followed by adding of sample protein (5μl/well)
• • Mix wells thoroughly with pipet.
• • Cover the plate and incubate at 37°C for 30 minutes.
• • Cool down the plate to RT.
• • Measure the absorbance at or near 562 nm in a plate reader.
• • Make a standard curve using data of standard protein. Calculate concentration of sample protein from the standard curve.