Determining the protein concentration of samples is a crucial requirement for most research studies. Since it is an application widely used in all laboratories, several kits and products are available to choose from. The BCA protein assay kit is one of the kits available today.
Pierce's BCA Protein Assay Kit is a quick and easy method for determining the concentration of proteins in whole lysates as well as in purified, column-separated protein fractions. Although the insert from the kit suggests that the kit could be used to study protein–protein interactions, measuring column fractions after chromatography, estimating the percent recovery of membrane proteins and other applications, we have frequently used the kit just to measure the protein concentrations in whole lysates prior to downstream applications such as Western blotting. We have used this kit innumerable times and have always had good and consistent results.
The protocol for the assay is very simple and easy to perform. The kit comes with two reagents, reagent A and reagent B, which need to be mixed at 50:1 ratio to make the working reagent and should be used immediately. The protein to be assayed and a standard protein, such as bovine serum albumin (BSA) in a convenient range, are incubated with the working reagent for 30 min and read at 562 nm. It is convenient that the kit also comes with BSA at 2 mg/ml concentration for use as a standard. Another convenient feature of this kit is that the presence of other substances in the lysates does not interfere with the reaction. The product page on Thermo Scientific's website has a list of substances that are compatible with this kit.
The principle of this kit is based on two reactions. In the first reaction, the protein from the lysate is in an alkaline medium and chelates the Cu2+ ions and reduces them to Cu1+ ions, forming a pale blue complex. In the second step, the chelated cuprous ions combine with bicinchoninic acid (BCA) to form a strong purple colored complex. The intensity of the color is proportional to the concentration of the protein and can be read colorimetrically at 562 nm. Although the presence of certain specific amino acids in the protein, such as lysine, cystein, tyrosine and tryptophan are crucial for the color formation, the composition of the protein doesn’t appear to alter the sensitivity of the assay.
In summary, this kit contains reagents that are stable for 1 year; the assay is fast, accurate and precise; the reaction tolerates the presence of other substances in the lysates; the end point is an easily detectable colored compound and, in addition, very small quantities of lysates can be used in the microplate format. Considering all of the above features, I can say that Pierce's BCA Protein Assay Kit can be used as the choice kit for protein concentration determination in most applications.
Scientist
Children’s Research Center of Michigan
Children’s Hospital of Michigan