RC-DC Protein Assay From Bio-Rad

Protein estimation is one of the most basic and crucial experiments in biochemistry. Accurate estimation is necessary for assessing the levels of specific protein for Western blotting which has a sensitivity range from nano to picograms of protein. Most protein estimation is based either on the Lowry or Bradford method. These two methods have been modified and kits are available allowing one to select the proper kit for the type of lysis solution (containing interfering agents) or protein solution one has.

Bio-Rad has a line of protein estimation kits, namely: Bradford Assay, Bio-Rad Protein Assay, DC Assay and RC DC Assay. The first two kits are based on the Bradford method and the other two are based on the Lowry method of protein estimation. The selection of these kits depends on the concentration of specific chemicals in the sample, the availability of filters (for detection) in the lab and the amount of protein available for estimation. The Bio-Rad Protein Assay can be used with reducing agents, but not with detergents. The DC Assay can be used with detergents, but cannot be used for samples containing reducing agents. However, the RC DC Assay can be used for samples containing both reducing agents as well as detergents, which are the major interfering agents in protein estimation. Hence, this assay is ideal for protein samples for 2-dimensional electrophoresis (2DE).

The RC DC Assay is based on the principle of Lowry estimation, where protein reacts with alkaline copper and subsequently reduces the folin reagent and leads to color development. This color development is basically because of changes to tyrosine and tryptophan and to a lesser extent, cysteine and histidine. The absorbance of the final product can be measured at 750 nm.

The RC DC Assay Kit comes with Reagent A (which contains alkaline copper tartarate solution), Reagent B (which contains a dilute folin reagent), Reagent S, RC reagent I, RC reagent II, and either bovine serum albumin (BSA) or bovine gamma globulin. (RC DC Assay Kit II comes with BSA; RC DC Assay Kit I comes with gamma globulin.) I prefer BSA as we have been using BSA for more than 6 years. The assay can be performed with 5 ml tubes or 1.5 ml microfuge tubes. I prefer to perform the assay in 1.5 ml microfuge tubes as it saves lot of reagent.

First, 5 ul of Reagent S is added to 250 ul of Reagent A; each tube requires 127 ul of this reagent, which is called A’. Three to five dilutions of protein are prepared (0.2-1.5 mg/ml) and 25 ul of protein standards are added to the microfuge tubes. 125 ul of RC Reagent 1 is added followed by 125 ul of RC Reagent II. These reagents precipitate the protein. After vortexing the tubes, they are centrifuged at 15,000g for 5 minutes. The supernatant is discarded completely (which contains soluble interfering agents) and 127 ul of A’ is added. After incubation at room temperature for 5 minutes, 1 ml of Reagent B is added to each tube. Tubes are vortexed and after a 15 minute incubation, readings are taken at 750 nm.

The best part of the assay is the ease of performing the estimation. Previously, it would take 3½-4 hours to estimate the protein (when we performed TCA precipitation protocol), now it just takes 1-1½ hour. When I started the estimation for the first time, the assay showed an odd result: all the tubes went out of range. This was because the reducing agent was still present. The troubleshooting guide proved very helpful in finding out the problem and now I give one extra wash with RC Reagent I and RC Reagent II (basically, a repetition of two steps to remove residual reducing agents) and the readings are beautiful. The only disadvantage of the assay is the cost of the kit.

I would happily recommend this kit for all who perform 2DE as well as for anyone whose samples contain interfering, soluble chemicals.