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What Are the Different Methods of Protein Concentration Determination?

In one method of protein concentration determination, a colored dye that binds to protein is added to a protein solution.
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  • Written By: Aaron Lin
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  • Last Modified Date: 17 September 2014
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There are hundreds of different methods of protein concentration determination. The incredible diversity in the types of protein solutions that biochemists analyze is why there is no single universal method that works for every type of protein solution. The most common protein assays are the Bradford assay, the Lowry assay and the bicinchoninic acid assay. Nevertheless, myriad variations have been developed as needed to work around any potential chemical incompatibilities between the protein solution and the reagents that are being used in the assay.

Generally speaking, there are two main categories of assays for protein concentration determination. In the first group of methods, a colorful or fluorescent dye is added to a protein solution, and it binds specifically to protein. The bound dye has an unique absorption wavelength that is proportional to the amount of protein. By using a spectrometer, it becomes possible to estimate the concentration of protein.

The second group of assays involves adding copper(II) ions to a solution of protein, where these ions are reduced to copper(I) ions. These reduced ions are then able to form colorful complexes by binding to proteins. By measuring the absorbances at their unique wavelengths, the protein concentrations can likewise be inferred.

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One of the most popular methods of protein concentration determination is the Bradford assay. In this assay, a red dye called coomassie brilliant blue is added to a protein solution under acidic conditions. As this dye binds to protein, it form a permanent blue complex with a characteristic absorbance at 595 nanometers.

Despite the general versatility of the Bradford assay, it is incompatible with some protein solutions. In particular, the Bradford assay is disrupted by the presence of sodium dodecyl sulfate (SDS), a detergent that is commonly used to purify proteins and break down cells by lysis. This detergent interferes with the binding of the dye to proteins, which results in an unreliable and inaccurate absorption reading. Other types of methods, then, must be used when SDS is present.

Another series of protein assays has been developed, and they all involve a variation of the Biuret test. In this reaction, a protein is combined with an aqueous base and copper(II) ions. These ions are reduced and then chelated by protein to form colorful complexes. Two assays that use this test are the Lowry assay and the bicinchoninic acid assay.

With the Lowry assay, a Folin-Ciocalteu reagent is added to the Biuret test. The Folin-Ciocalteu reagent oxidizes aromatic residues, particularly tryptophan, and helps the complex absorb strongly at 750 nanometers. The bicinchoninic acid assay, on the other hand, involves adding bicinchoninic acid to the Biuret test. After a brief incubation at about 104° Fahrenheit (40° Celsius), two equivalents of acid and the peptide bonds of the protein chelate a single copper(I) ion. The result is a complex that absorbs strongly at 562 nanometers.

When selecting a method for protein concentration determination, it is important for one to consider the different chemical functional groups present in the solution. The presence of certain amino acid side chains, disulfide bonds and cofactors can make the protein concentration determination wildly inaccurate. It is often necessary for one to consider not only the proteins but also other reagents and buffers, such as reducing agents and detergents. The ideal method will be chemically compatible as well as be reliable, cheap and simple to set up.

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