The BCA Protein Assay Kit from Thermo Scientific Pierce (Product No. 23225) is a detergent-compatible, bicinchoninic acid formulation for the colorimetric detection and quantification of total protein. This method utilizes bicinchoninic acid (BCA) as the detection reagent for Cu+1, which is formed when Cu+2 is reduced by protein in an alkaline environment. A purple-colored reaction product is formed by the chelation of two molecules of BCA with one cuprous ion (Cu+1). In the absence of protein, the reagent maintains its green color. This water-soluble complex exhibits a strong absorbance at 562 nm; the absorbance increases is linear with increasing protein concentrations.
According to the manufacturer’s protocol, the sensitivity range of this assay is 20 µg/ml-2000 µg/ml. The kit comes with all reagents needed to perform the assay, including BSA as standard control. The kit is sufficient to perform 500 test tube or 5,000 microplate assays. Users need to supply test tubes, 96-well microplates or cuvettes and a spectrophotometer. The assay takes approximately an hour to complete if using the quick method or 2.5 hours to complete using the slow method. The difference in the methods is whether or not you incubate your samples at 37ºC (30 minute incubation) or room temperature (2 hour incubation). I have not performed the room temperature method so I do not know if it is more sensitive, less sensitive or about the same.
The major advantage I have found with using this kit is that it is compatible with a lot of different substances that you might want to place in the buffer; a list of compatible substances can be found on Pierce’s website. In order to do get the most accurate and sensitive readings, I have found that you need to dilute and prepare your BSA standards in the same buffer that your protein samples are stored in to allow for correct analysis.
The negative side to this protocol is that I hate performing dilution calculations. In order to perform the assay, you have to mix Reagent A with Reagent B to generate a working reagent (WR). The WR is what is then mixed with your protein sample. The protocol recommends a 50:1 (Reagent A:Reagent B) mixing ratio. This can be tricky to calculate when the total number of samples you are assaying is not directly divisible by 50. It’s just a nuisance; it would be really nice if Pierce could find some way to mix equal amounts of the two reagents to make it more simplistic.
Our lab has been extensively using the BCA Protein Assay Kit for the last year. In our hands, the kit has been an extremely sensitive and reliable colorimetric protein assay. Each of the commonly used total protein assay methods exhibits varying responses toward different proteins. These differences are related to amino acid sequence, pI, structure and the presence of certain side chains or prosthetic groups that can dramatically alter the protein assay’s response. Most protein assay methods utilize either BSA or immunoglobulin (IgG) as the standard against which the concentration of protein in the sample is determined. Pierce’s Albumin Standard (BSA) provides a consistent standard for estimating protein concentration.
Finally, I have not performed the microplate version of the assay because the manufacturer recommends different ratios of the reagent. Although this extends the life of the kit, the manufacturer does warn that the assay may not be as accurate as performing the test tube version.
Assistant Professor
Department of Biological Sciences
San Jose State University