The Pierce Coomassie Protein Assay Kit is used in our laboratory for quick quantification of total protein for prepared extracts from C. elegans
. This kit is sufficient for 630 test tube assays or 3800 microplate assays. It comes with 950 ml of Coomassie (Bradford) Protein Assay Reagent, which needs to be stored at 4º
C and 10 x 1 ml ampules of 2 mg/ml albumin (BSA) standard. The reagents should be warmed to room temperature before use, as cold reagent may affect the assay. Also, certain substances in protein extracts, such as ionic or nonionic detergents, may interfere with the assay.
Theoretically, Coomassie dye binds to proteins in an acidic medium, causing a shift in the absorption maximum from 465 nm to 595 nm. The color of the dye changes from brown to blue, enabling a good estimation of protein concentration using absorbance calculated from a standard curve (a new curve must be made for each assay because the color change is non-linear). To generate a standard curve, I make a series of dilutions (usually 6 dilutions) of the standard 2 mg/ml albumin and mix 3 ul of each dilution with 1 ml of the Coomassie Protein Assay Reagent. To determine the protein concentration of unknown protein extracts, I add between 1 and 10 ul, depending on the observable color change of the assay reagent. After blanking the spectrophotometer, absorbance is measured at 595 nm. In order to get the most accurate information, the absorbance of your unknown samples should fall between the OD measurements of your highest and lowest BSA standard dilutions.
In my research, I am looking at the levels of different proteins which are implicated in the regulation of aging using a variety of long-lived mutants. I need to measure protein concentration of my worm extracts in order to load similar amounts of protein per well for Westerns. The advantages of using this kit are that we don’t have to spend the time making the reagents ourselves, and the reagents have been quality tested. Also, the kit includes 2 mg/ml BSA capsules which we can use to standardize our assays.
As mentioned previously, this kit can be used for microplate assays as well, but I have not tried that method yet. The protocol that comes with the kit is very informative and easy to follow. I have not had any negative experiences with this assay kit.
Hee Chul Lee
University of Michigan Medical School
Coomassie (Bradford) Protein Assay Kit From Pierce
Reliable, consistent readings, sensitive and easy to follow manual.
The Bottom Line
The kit comes with good amount of Bradford dye and with BSA stock for standardization. Highly recommended.