The BCA assay is used to quantify protein concentration by using bicinchoninic acid to identify copper ions reduced by protein in a biuret reaction. The BCA protocol requires a working solution mixed with the sample; when protein is present, the reaction produces a purple color that absorbs light at 562 nm and is quantified with a spectrophotometer. The BCA assay is similar to other protein quantification assays such as Lowry or Bradford assays. However, the biuret reaction of the BCA assay occurs between the nitrogens on the peptide backbone and copper as well as nitrogens on the amino acid side chains. The fact that the peptide backbone participates in the reaction means that the BCA assay is more consistent between proteins and is less dependent upon amino acid composition. The BCA protocol is simple and quick. If the sample is heated to 37°C, then the incubation time is only 30 minutes, and the absorbance measurement takes only a few minutes. The BCA assay is an excellent method for quantifying total protein concentration after biomagnetic protein purification.
The BCA assay chemical reaction
Peptide bonds reduce copper (Cu2+ to Cu1+) in a two step reaction between the nitrogens of the peptide backbone and the copper in an alkaline environment. The peptide backbone is composed of amino nitrogen, alpha carbon, and carboxyl carbon of each amino acid (NCC) linked to create a pattern of NCCNCCNCC. The BCA assay requires a working solution of sodium hydroxide, copper sulfate, and sodium potassium tartrate in order for the biuret reaction to occur. Once the monovalent copper ion is released by the reaction, it is chelated by bicinchoninic acid to provide a quantitative readout for the assay.
Bicinchoninic acid sodium salt is the molecule responsible for the readout of the BCA assay; it is composed of two carboxylated quinoline rings. Two molecules of bicinchoninic acid chelate one copper ion to create a complex that turns purple and absorbs 562 nm light. The copper ion Cu1+ is only present when proteins are in the sample.
This process works well for proteins at concentrations between 20 µg/mL and 2000 µg/mL.
It is important to note that the biuret reaction occurs between nitrogens and copper. This means that buffers such as Tris will lead to false positive results. Additionally, other reducing agents in the sample can interfere with the reaction. Things like DTT, beta mercaptoethanol, and EDTA (a copper chelator) must be avoided.
BCA protocol stages
- prepare a standard curve of known protein concentration
- prepare mixture of sodium hydroxide, bicinchoninic acid, and sodium potassium tartrate. This is also known as the “working solution” in many commercial kits
- add working solution to sample solution in test tubes or in a multiwell plate (96-well is commonly used)
- incubate for 2 hours at room temperature or for 30 minutes at 37°C
- take a blank measurement on a spectrophotometer at 562 nm
- measure sample with a spectrophotometer at 562 nm and subtract blank
- compare absorbance values to standard curve to obtain protein concentration of the sample