The Micro BCA™ Protein Assay Kit from Pierce Biotechnology is a detergent-compatible, bicinchoninic acid formulation for the colorimetric detection and quantitation of total protein. An adaptation of the BCA™ Protein Assay Kit (Product No. 23225), the Micro BCA™ Protein Assay Kit has been optimized for use with dilute protein samples (0.5-2.0 µg/ml). 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). This water-soluble complex exhibits a strong absorbance at 562 nm that is linear with increasing protein concentrations.
Our lab has been extensively using the Micro 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. However, individual proteins, including BSA and IgG, differ slightly in their color responses in the Micro BCA™ Assay. Therefore, the standard curve should be prepared from a pure sample of the target protein to be measured in cases of great accuracy. Nevertheless, based on our experience the Micro BCA™ Protein Assay Kit is one of the best protein assay kits available on the market.
According to the manufacturer’s instructions, the kit has sufficient reagents for 480 tube assays or 3,200 microplate assays. In our experiments we always used the kit for a test tube procedure. Preparation of standards and working reagents are very easy to make based on the guidelines given. I would recommend preparing sufficient volume of working reagents based on the number of samples to be analyzed.
The assay may initially take several hours but once the protocol is mastered, the results will be available in a couple of hours. After preparing the standards and working reagents and mixing them with the tested sample, you will need to cover the tubes and incubate at 600C in a water bath for 1 hour. After incubation, the tubes are cooled at room temperature and the absorbance of all the samples are measured with a spectrophotometer set to 562 nm. The procedure for microplates differs in that you will need much less standard and working reagents to carry out your experiment and incubation at 370C for 2 hours.
In conclusion, the Micro BCA™ Protein Assay Kit is a best-fit companion for the colorimetric detection and quantitation of total protein. It is a sensitive assay that can be used in either a test tube or microplate format. Its consistent results and fairly easy-to-use protocol makes it an asset in any research lab dealing with quantitation of total protein. I would have no problem recommending this product!
Alisher R. Dadabayev, MD
Postdoctoral Research Associate
Laboratory of Immunology
Department of Internal Medicine
TTUHSC, Amarillo