BradfordMethode

BradfordMethode, often called the Bradford protein assay, is a colorimetric method for quantifying total protein in a sample. It was developed by Marion M. Bradford in 1976 and uses the dye Coomassie Brilliant Blue G-250. In its acidic form the dye binds to proteins and shifts its absorbance from about 465 nm to 595 nm, producing a blue color. The intensity of the blue is proportional to the protein concentration within a certain range, allowing measurement via a standard curve.

Principle and execution

The method relies on the interaction between the dye and protein, which is enhanced by basic amino acids, particularly arginine, and to a lesser extent by other residues. A small sample is mixed with Bradford dye reagent, incubated briefly, and the absorbance is measured at 595 nm. A standard curve is created using a known protein, commonly bovine serum albumin, to determine the concentration of unknown samples. The typical working range depends on the protein and conditions but is commonly in the tens to hundreds of micrograms per milliliter.

Advantages and limitations

The Bradford assay is fast, simple, inexpensive, and requires only small sample volumes. It is compatible with many common buffers and is widely used in routine protein quantification. However, its accuracy can be affected by the protein’s amino acid composition, and the method is susceptible to interference from certain substances, such as strong detergents, reducing agents, and high salt or phosphate buffers. Therefore, calibration with an appropriate standard and awareness of sample composition are important for reliable results.

See also

Coomassie Brilliant Blue, Lowry method, BCA assay.