Bradford protein assay
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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.
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[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 changed and stabilized into coomassie blue by the binding of protein. Two types of bond interaction take place here, the red form of coomassie dye first donates its free proton to the ionizable groups on protein, which causes a disruption of protein's native state, and consequently exposes its hydrophobic pockets. The exposed hydrophobic pockets on the protein chain will bind non-covalently to the non-polar region of the dye via van der Waals force, this will position the positive amine groups to proximity with the negative charge of dye, and the bond is further strengthened by the ionic interaction between the two. Binding of the protein stablized the blue form of coomassie dye, and the amount of the complex present in solution is a measure for the protein concentration by use of 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 dye 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. Common detergent such as SDS, could be found in protein extracts because it is used in lysing cells by disrupting its membrane lipid bilayer. While other detergents interfere with the assay at high concentration, interference caused by SDS is of two different modes, and each occurred at a different concentration; for SDS concentration 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 and pre-empting the protein binding sites for dye reagent, which caused an underestimation of protein concentration in solution. For SDS concentration above CMC, it associated strongly with the green form of coomassie dye, and causes the equilibrium to shift in producing more of the green form, which will increase the absorbance at 595nm independent of protein presence. Other interference may come from the buffer used when preparing the protein sample. High concentration of buffer will cause an overestimated protein concentration due to depletion of free protons from the solution by conjugate base from buffer. This will not be a problem if 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 tissue 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.
[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 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.
[edit] 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 assay
The assay kit provided by SIGMA is linear for concentrations up to 1.4 mg/ml
[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 (1995) Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal. Biochem. 236:302-8
[edit] External links
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