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Bradford protein assay
Additional recommended knowledge
The Bradford assay, a colorimetric protein assay, is based on an absorbance shift in the dye Coomassie when the previously red form commassie 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 disrupture 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 anionic (unbound) forms are green and red. 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 CDC, 0.00333%W/V to 0.0667%) in a coomassie dy solution, it tends to bind well with protein and pre-emptying the protein binding sites for dye reagent, which caused an underestimation of protein concentration in solution. For SDS concentration higher 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 independet 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.
The Bradford assay is linear over a short range, typically from 2ug/ml to 120ug/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.
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.
Alternative protein assays include
The assay kit provided by SIGMA is linear for concentrations up to 1.4mg/ml
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Bradford_protein_assay". A list of authors is available in Wikipedia.|