Bradford test

from Wikipedia, the free encyclopedia

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

BSA standard series from 0 µg left to 12 µg right, mixed with Coomassie blue, in a 96-well plate

principle

The triphenylmethane dye Coomassie brilliant blue G-250 (CBBG) forms complexes with cationic and non-polar side chains of proteins in acidic solution . The unbound (cationic), red form of the dye has a maximum at 470 nm in the absorption spectrum . The complex formation with proteins stabilizes the dye in its blue, unprotonated, anionic sulfonate form 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 can be measured photometrically due to the formation of the complex with high sensitivity to the free color reagent and is a measure of the protein concentration of the solution.

The extent of the color reaction depends on the protein; a calibration is therefore necessary to determine the concentration of a certain protein . If this is not available or if the concentration of a protein mixture is to be determined, standard proteins are used for calibration (e.g. chymotrypsin , lysozyme or BSA ). Depending on the composition of the protein mixture, different results can be obtained for the same amount of protein. Thus the Bradford determination is imprecise here. Their advantages are their high sensitivity and the simple and quick implementation.

Limit of quantitation

The limit of quantification of the microtest is 1–20 µg / ml, the macrotest 20–200 µg / ml protein.

Interfering substances

The Bradford test is already supported by low concentrations of detergents such as B. SDS disturbed. These also bind to the protein and compete with the dye for binding sites or displace it from the protein, which prevents the color reaction. In contrast, chaotropic compounds such as B. guanidinium chloride , reducing agents such as DTT ( dithiothreitol ) or chelators do not affect the result. If detergents cannot be eliminated from the protein samples, other evidence, e.g. B. the BCA test - which is insensitive to detergents and, like the Lowry test, has a lower detection limit - can be used.

advantages

  • Fast and inexpensive
  • Very sensitive
  • The dye-protein complex is stable for just under an hour

disadvantage

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

Individual evidence

  1. Bradford, MM (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. In: Anal. Biochem. Vol. 72, pp. 248-254. PMID 942051 doi: 10.1016 / 0003-2697 (76) 90527-3 PDF .
  2. Compton, SJ, Jones, CG: Mechanism of dye response and interference in the Bradford protein assay. In: Anal. Biochem. 151: 369-374 (1985). PMID 4096375 doi: 10.1016 / 0003-2697 (85) 90190-3 .
  3. ^ Bio-Rad Laboratories: Quick Start Bradford Assay Instruction Manual . P. 7.

See also

Web links