Bradford assay reagent in a ready-to-use formulation that allows for total protein determination in seconds.

The Thermo Scientific Pierce Coomassie (Bradford) Protein Assay Kit is a ready-to-use formulation of the popular assay reagent originally described by Bradford in 1976. 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.

Highlights:

• Ready-to-use dye-binding reagent formulation
• Fast (almost immediate) color development; measure at 595 nm
• Compatible with reducing sugars, reducing substances and thiols
• Refrigerated reagent is stable for 1 year
• Useful for determining protein concentration from 100-1,500 µg/ml
• Micro method determines protein concentration in the range of 1-25 µg/ml
• Adaptable to microplates

Standard curves. Typical standard curves for bovine serum albumin (BSA) and bovine gamma globulin (BGG) in the Pierce Coomassie Protein Assay. The assay kit (#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 465 nm) to the blue form of the dye (absorbance maximum at 610 nm). The difference between the two forms of the dye is greatest at 595 nm, 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 575 nm and 615 nm. At the two extremes (575 nm and 615 nm) there is a loss of about 10% in the measured amount of color (absorbance) compared to that obtained at 595 nm.

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 3,000 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 595 nm 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.

References:

1. Bradford, M. (1976). Anal. Biochem. 72, 248-254.
2. VanKley, H. and Hale, S.M. (1977). Anal. Biochem. 81, 485-487.
3. Messenger, M.M., et.al. (2002). J. Biol. Chem 277, 23054-23064.

Related Links:
Protein:Protein Variation of Protein Assays
Protein Assay Selection Guide

Protein Methods Library:
Overview of Protein Assays
Chemistry of Protein Assays
Protein Assay Data Analysis

Compatible Instrumentation:
BioMate 3 UV-Vis Spectrophotometer
BioMate 5 UV-Vis Spectrophotometer
NanoDrop 2000c UV/Vis Spectrophotometer
NanoDrop 8000 UV/Vis 8 Sample Spectrophotometer

Related Products:
BSA & BGG Protein Assay Standard Sets
Coomassie Plus (Bradford) Protein Assay Kit
Pierce 660 nm Protein Assay
BCA Protein Assay Kit