Bradford protein assay

The Bradford assay is a photometric method for the quantitative determination of protein concentrations in the range up to micrograms per milliliter. It is named after the American biochemist Marion M. Bradford.

Principle

The triphenylmethane dye Coomassie Brilliant Blue G -250 ( CBBG ) forms in acid solution with cationic and non-polar side chains of proteins complexes. The unbound (cationic ), red form of the dye having the absorption spectrum has a maximum at 470 nm Due to the complex formation with proteins of the dye is stabilized in its blue, unprotonated, anionic sulfonate and the absorption maximum shifts to 595 nm, since the extinction coefficient of the dye -protein complex is also much higher than that of the free dye, the increase in absorbance at 595 nm due to the formation of the complex with a high sensitivity against the free color reagent can be measured photometrically, and is a measure of the protein concentration of the solution.

The measure of the color reaction is dependent on the protein; Therefore, a calibration is necessary for determining the concentration of a particular protein. If this is not available or if the concentration of a protein mixture are determined, standard proteins used for calibration (eg, chymotrypsin, lysozyme or BSA). This can be obtained depending on the composition of the protein mixture of equal amounts of protein different results. Thus, the Bradford determination is inaccurate here. Its advantages are its high sensitivity and the quick and easy implementation.

Limit of

The detection limit of the assay is 1-20 micro g / ml, the macro tests 20-200 ug / ml protein.

Interfering Substances

Salts at physiological concentrations and sucrose have no influence on the result. In contrast disturb detergents such as SDS, denaturing agents such as urea, guanidinium chloride or thiourea, and reducing agents such as DTT ( dithiothreitol ). This susceptibility is a further disadvantage of the Bradford assay, since these substances are routinely used in the protein chemistry. Can not be eliminated from the protein samples them, it is possible to calibrate the tests and the protein Policy - carried out in the presence of a fixed concentration of interfering substance - within certain limits. Here, however, it loses its sensitivity.

Benefits

• Quick and inexpensive
• Very sensitive
• Dye -protein complex is stable for about an hour

Disadvantages

• Calibration necessary
• Susceptible to interference from protein chemical reagents
• Non-linear standard curve over a wide range
• The sensitivity for different proteins can spread widely, making the choice of standards is important. The method is an accurate quantification can only be of limited use.