DC Protein Assay From Bio-Rad

The DC Protein Assay is one of many colormetric assays commercially available for protein concentration quantification. This assay is based upon a simple reaction whereby the protein within a solution reacts with a Folin reagent and an alkaline copper tartate solution. The outcome of this reaction is the formation of a blue product (the more protein, the darker the blue). This is ideally read between 650 nm and 750 nm (though it can be read over a spectrum starting at 405 nm). The fore-mentioned redox reaction is reliant on the ability of certain amino acids (especially tryptophan and tyrosine) to reduce the Folin reagent provided in the kit. This leads to the formation of a variety of reduced species, all of which are blue in color.

This assay is similar to that of the established Lowry assay but it has two improvements: (1) This assay only takes 15 minutes to reach 95% of maximum color development. (2) The color produced in the reaction fades slowly (less than 5% after 60 minutes and less that 10% after 120 minutes) and hence the result does not have to be read immediately after development.

Depending on the amount of protein sample you have, you will either use the assay in the standard or microplate format. The standard assay requires 100 ul of standards or samples per tube whereas the microplate assay is far more economical, requiring only 5 ul of standard or samples per well. For the microplate protocol, first pipette the following (in this order) into each well (in duplicate or triplicate): Standards/samples (5 ul) plus a blank (lysis buffer), 25 ul reagent A (mix), 200 ul reagent B (mix). If your lysis buffer contains detergent, add 20 ul of reagent S to each ml of reagent A. Shake the plate vigorously for 15 minutes at room temperature. Read at 750nm. For the standard protocol, pipette the following into clean, dry test tubes: Standards/samples 100 ul, 500 ul reagent A (vortex), 4 ml reagent B (vortex). Shake the tubes vigorously for 15 minutes at room temperature and read at 750nm.

This kit can be bought with or without protein standards, with the option of ã-globulin or bovine serum albumin (BSA), both of which give consistent and comparable results. Alternatively, the researcher can simply make up standards cheaply using your lysis buffer to dilute a protein such as BSA. This assay has the advantage that it is compatible with many reagents commonly used in lysis and elution buffers, such as SDS, EDTA and triton-X-100, so you can use whole cell protein extracts or purified protein. Additionally, this assay can be used on buffers containing the common reducing agents DTT and beta-mercaptoethanol. Many protein assay kits are not compatible with these reagents, but please bear in mind that this compatibility has a limited threshold, so this kit should not be used in the presence of high concentrations of reducing agents.

I have used this kit on whole cell protein extracts from mammary carcinoma cell lines. The protein extracted is subsequently used in Western blots so accurate quantification is essential. Because my yields of protein from different cell lines are quite variable and the cells are lysed in a small volume of lysis buffer (usually less than 200 ul), I use the microplate protocol as to not ‘waste’ sample.

I have found this assay to be quick and very reliable. It is two times faster than the common BCA Assay (Pierce) and when using the microplate technique, only requires a fifth of the 25 ul sample volume typically required for the BCA.