Bradford protein assay Information & Bradford protein assay Links at HealthHaven.com

This article is about the scientific procedure. For other uses, see Bradford (disambiguation).

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.

[edit] 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 electron 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 blue 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.

[edit] 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.

[edit] 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 Bradford assay linearizes by measuring the ratio of the absorbances, 595 over 450 nm. This modified Bradford assay is approximately 10 times more sensitive than the conventional one. (Zor & Selinger, 1995)

[edit] Alternative assays

Alternative protein assays include

[edit] 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 (1996) Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal. Biochem. 236:302-8

[edit] External links