The BCA Protein Assay Kit provided by Pierce Biotechnology (now part of Thermo Fisher Scientific), is a comprehensive and robust protein quantification assay based on the colorimetric detection of copper ions which are chelated with proteins. Any peptide sequence containing 3 or more amino acids (tri-peptides or longer) can reduce Cu
2+ cations to Cu
1+ in an alkaline medium which results in the formation of a protein-copper chelate that has a distinct blue color. A single Cu
1+ ion can form a complex with 4 or 6 surrounding peptide bonds. An increasing number of peptide bonds results in the formation of more blue-colored chelates; therefore, the amount of protein is directly proportional to the intensity of the blue color observed. In addition, two molecules of BCA (bicinchoninic acid) react with a single Cu
1+ ion to form a purple-colored chelate. This complex is water soluble and exhibits strong linear absorbance with increasing protein concentration (more peptide-copper chelates) at 562 nm.
The BCA Protein Assay is, therefore, performed as a two-step protocol. BSA (bovine serum albumin) protein standard dilutions (provided with the kit) along with the protein samples to be analyzed are added to a 96-well microplate. Two reagents, provided with the kit, are mixed together and immediately ready for use. The first reagent, Reagent A, contains the BCA in a sodium bicarbonate and sodium hydroxide buffer and the second reagent, Reagent B, contains the Cu2+ ions in cupric sulfate form. This mix is added to the protein samples. I find it easier to load the microplate with the reagent mix (200 ul per well) and then add the protein samples (25 ul each) sequentially. It is definitely a good idea to make the reagent mix in excess because if you aim for 26 wells and prepare the mix for as many wells, you invariably find yourself running short near the end!
The reaction is very fast and within minutes, a difference in color is observed between samples with different protein concentrations. This is a good visual guide to give yourself a pat on the back if the color intensities match your concentration predictions! Of course, this is not a quantitative assessment and the samples do require an incubation period of 30 minutes at 37°C. This time period and temperature is recommended by the manufacturer although it can be done at room temperature for a longer incubation period (1.5 or 2 hours). Theoretically, the color will continue to develop with decreasing intensity, and the literature reports that about 30 minutes at 37°C is optimal. I’ve tried 20 minute incubation periods at 33°C and observed negligible differences in absorbance readings.
I use the BCA Protein Assay Kit routinely to quantify crude or fractionated protein extracts from various plant seedling tissues or cell cultures. I keep the standard dilutions as pre-prepared stocks at –20°C and re-use these for different experimental replicates without any loss in standard curve accuracy. I then take absorbance measurements using a Tecan Infinite 200 Quad-4 spectrophotometer (also reviewed on Biocompare). All in all, the BCA Protein Assay is a very robust kit and I recommend it to anyone, especially those involved in high throughput protein analysis because of the rapid quantification option.
Research Fellow
Plant Immunity Research Team
RIKEN Plant Science Center