The Thermo Scientific Pierce Coomassie Protein Assay Kit is a ready-to-use, stable formulation of the traditional Bradford assay reagent to measure (A595nm) total protein concentration compared to a protein standard.
The Pierce Coomassie Protein Assay Kit is a ready-to-use formulation of the popular assay reagent originally described by Bradford in 1976. When mixed with a protein solution, the acidic coomassie-dye reagent changes color from brown to blue in proportion the amount of protein present in the sample. Protein determinations are made by comparison to the color response of protein assay standards, usually prepared as a series of known dilutions of bovine serum albumin (BSA) or bovine gamma globulin (BGG). The kit includes Coomassie Protein Assay Reagent and a package of Albumin Standard Ampules. The simple procedure is adaptable to nearly any volume scale, including test tubes, cuvettes and microplates.
- Bradford reagent – stable, ready-to-use kit of the classical Bradford assay reagent
- Colorimetric – measure with a standard spectrophotometer or plate reader (595nm)
- Easy to use – single reagent; no working reagent preparation required
- Fast – almost immediate color development; add, mix and read results
- Broad range – detects protein concentration in the range 1 to 1500µg/mL
- Flexible – microplate and cuvette protocols provided with the instructions and adaptable to several target working ranges
|Standard curves. Typical standard curves for bovine serum albumin (BSA) and bovine gamma globulin (BGG) in the Pierce Coomassie Protein Assay. The assay kit (Part No. 23200) includes ampules of Albumin Standard.
How the Coomassie (Bradford) Assay Detects Protein:
Use of coomassie G-250 dye in a colorimetric reagent for the detection and quantitation of total protein was first described by Dr. Marion Bradford in 1976. In the acidic environment of the reagent, protein binds to the coomassie dye. This results in a spectral shift from the reddish/brown form of the dye (absorbance maximum at 465nm) to the blue form of the dye (absorbance maximum at 610nm). The difference between the two forms of the dye is greatest at 595nm, so that is the optimal wavelength to measure the blue color from the coomassie dye-protein complex. If desired, the blue color can be measured at any wavelength between 575nm and 615nm. At the two extremes (575 nm and 615nm) there is a loss of about 10% in the measured amount of color (absorbance) compared to that obtained at 595nm.
Development of color in coomassie dye-based (Bradford) protein assays has been associated with the presence of certain basic amino acids (primarily arginine, lysine and histidine) in the protein. Van der Waals forces and hydrophobic interactions also participate in the binding of the dye by protein. The number of Coomassie dye ligands bound to each protein molecule is approximately proportional to the number of positive charges found on the protein. Free amino acids, peptides and low molecular weight proteins do not produce color with coomassie dye reagents. In general, the mass of a peptide or protein must be at least 3000 daltons to be assayed with this reagent. The assay is performed at room temperature and no special equipment is required. Simply add the sample to the tube containing reagent and the resultant blue color is measured at 595nm following a short room-temperature incubation. The coomassie dye containing protein assay is compatible with most salts, solvents, buffers, thiols, reducing substances and metal chelating agents encountered in protein samples.
For more information, see the article "Chemistry of Protein Assays" in the Protein Methods Library.
- Bradford, M. (1976). Anal. Biochem. 72, 248-254.
- VanKley, H. and Hale, S.M. (1977). Anal. Biochem. 81, 485-487.
- Messenger, M.M., et.al. (2002). J. Biol. Chem 277, 23054-23064.