Bradford protein assay

The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. It is subjective, i.e. dependent on the amino acid composition of the measured protein.

Principle

The Bradford assay, a colorimetric protein assay, is based on an absorbance shift in the dye Coomassie when the previously red form coomassie reagent changes and stabilizes into coomassie blue by the binding of protein. During the formation of this complex, two types of bond interaction take place: the red form of coomassie dye first donates its free proton to the ionizable groups on the protein, which causes a disruption of the protein's native state, consequently exposing its hydrophobic pockets. These pockets on the protein's tertiary structure bind non-covalently to the non-polar region of the dye via van der Waals forces, positioning the positive amine groups in proximity with the negative charge of the dye. The bond is further strengthened by the ionic interaction between the two. Binding of the protein stabilizes the blue form of coomassie dye, thus the amount of complex present in solution is a measure for the protein concentration by use of an absorbance reading.

The (bound) form of the dye has an absorption spectrum maximum historically held to be at 595 nm. The cationic (unbound) forms are green or red while binding of the dye to protein stabilizes the blue anionic form. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample.

Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemicals that may be present in protein samples. An exception of note is elevated concentrations of detergent. Sodium dodecyl sulfate (SDS), a common detergent, could be found in protein extracts because it is used in lysing cells by disrupting the membrane lipid bilayer. While other detergents interfere with the assay at high concentration, interference caused by SDS is of two different modes, and each occurs at a different concentration. For SDS concentrations below critical micelle concentration (known as CMC, 0.00333%W/V to 0.0667%) in a coomassie dye solution, it tends to bind well with protein, inhibiting the protein binding sites for dye reagent. This can cause underestimations of protein concentration in solution. In SDS concentrations above CMC, the detergent associates strongly with the green form of coomassie dye, causing the equilibrium to shift, thereby producing more of the green form, which causes an increase in the absorbance at 595 nm independent of protein presence. Other interference may come from the buffer used when preparing the protein sample. A high concentration of buffer will cause an overestimated protein concentration due to depletion of free protons from the solution by conjugate base from the buffer. This will not be a problem if a low concentration of protein (subsequently the buffer) is used.

Disadvantages

The Bradford assay is linear over a short range, typically from 2 µg/ml to 120 µg/ml, often making dilutions of a sample necessary before analysis.

Because the Bradford assay essentially measures the amount of arginine and hydrophobic amino acid residues, the amino acid composition can alter the concentration-absorbance curve depending on the percentage of arginine or hydrophobic amino acids in each protein. It is therefore necessary to use a standard (e.g. BSA-- Bovine Serum Albumin) whose protein closely matches the measured protein in composition, but systematic error(different amino acid composition for all proteins) should be taken into account when performing the assay.

Modified Bradford procedure

Much of the non linearity stems from the equilibrium between two different forms of the dye which is perturbed by adding the protein. The nonlinearity issue of the Bradford assay may be solved by measuring the ratio of OD at 595 to 450 nm. This modified Bradford assay is approximately 10 times more sensitive and stable than the conventional one. Fact|date=July 2008

Alternative assays

Alternative protein assays include
* UV spectroscopy
* Biuret protein assay
* Lowry protein assay
* Bicinchoninic acid protein assay
* Amido black protein assay
* o-phthalaldehyde protein assayThe assay kit provided by SIGMA is linear for concentrations up to 1.4 mg/ml

References

* Bradford, M. M. (1976) "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding". Anal. Biochem. 72:248-254.
* Zor, T. and Selinger, Z (1995) "Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies". Anal. Biochem. 236:302-8

External links

* [http://www.bio-rad.com Bradford Assay Maker]
* [http://www.histosoft.com/services/background/ba/baback.html Bradford Assay Background]