The DC Protein Assay is a colorometric assay for the determination of protein concentration from Bio-Rad. Like other protein assays by Bio-Rad, the DC assay is based on established techniques, in this case the Lowry assay which dates back to 1951. There are two steps to this assay – first, copper reacts with protein in an alkaline environment and second, the copper-treated protein reduces Folin reagent to develop a blue color. Peparation of cell extracts in the presence of detergents, such as Triton X-100, is an extremely common procedure, however, detergents often interfere with other assays of protein concentration. This product is specifically designed for the determination of protein concentrations in samples that have been solublized in detergents.
In practice, the assay procedure is very simple. Reagent A is an alkaline copper tartrate solution. If detergents are present in lysis buffers, an extra reagent is initially added to A, but this is omitted if detergents are not present. Sample and standards are mixed with reagent A and then reagent B (Folin reagent) is added and mixed. The assay is obviously applicable to both cuvette and microplate assays if the indicated ratios of sample/standard and reagents are adhered to. Absorbance values can be read in 15 minutes, at 750nm, and protein concentration is determined with reference to a calibration curve. As with all protein assays, calibrations vary slightly depending on the ‘standard protein’ used (e.g. BSA, IgA, ribonuclease A) and so consistency is recommended.
The assay is improved over the original Lowry assay as color development is complete within 15 minutes and once developed, color is essentially stable for up to one hour. In addition to speed and stability, this assay has the advantage, as previously mentioned, of insensitivity to many detergents. A list of detergents is given that have been tested with the DC Protein assay and found to be compatible. This list includes 10 % SDS, 1 % Triton X-100, 1 % Tween 20 and 2 % NP-40. Further, other chemicals such as 0.05 % Na Azide, 0.4 M guanidine HCl and 1 mM DTT have also been tested and found not to interfere with the assay. As such, the assay is unaffected by many chemicals that are commonly included in cell lysates and other protein samples. There are, however, caveats: combinations of detergents may not be compatible and certain chemicals, such as 2-mercptoethanol, are incompatible. As such, it is recommended that the standard curve be set up in the same buffer as samples are prepared in.
Peter Haggie, Ph.D.
University of California, San Francisco