There are various methods for determining protein concentration, but no universal method due to the diversity of protein solutions. The most common assays involve adding a dye or copper ions to the solution and measuring absorbances. The Bradford assay is popular but incompatible with SDS. Other tests, such as the Lowry and bicinchoninic acid assays, use variations of the Biuret test. It’s important to consider the chemical groups present in the solution when selecting a method.
There are hundreds of different methods for determining protein concentration. The incredible diversity in the types of protein solutions analyzed by biochemists 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. However, a myriad of necessary variations have been developed to circumvent any potential chemical incompatibilities between the protein solution and the reagents used in the assay.
In general, there are two main categories of assays for determining protein concentration. In the first group of methods, a colored or fluorescent dye is added to a protein solution and specifically binds to the proteins. The bound dye has a unique absorption wavelength which is proportional to the amount of protein. Using a spectrometer, it becomes possible to estimate the protein concentration.
The second group of tests involves the addition of copper(II) ions to a protein solution, where these ions are reduced to copper(I) ions. These reduced ions are then able to form colored complexes by binding to proteins. By measuring absorbances at their unique wavelengths, protein concentrations can also be inferred.
One of the most popular methods for determining protein concentration 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 proteins, it forms 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. Notably, the Bradford assay is disrupted by the presence of sodium dodecyl sulfate (SDS), a commonly used detergent to purify proteins and break down cells by lysis. This cleaner 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 set of protein tests has been developed and 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 proteins to form colored 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. Folin-Ciocalteu reagent oxidizes aromatic residues, especially tryptophan, and helps the complex absorb strongly at 750 nanometers. The dosage of bicinchoninic acid, on the other hand, involves the addition of bicinchoninic acid to the Biuret test. After a brief incubation at approximately 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 strongly absorbs at 562 nanometers.
When selecting a method for determining protein concentration, it is important to consider the different chemical functional groups present in the solution. The presence of some amino acid side chains, disulfide bonds, and cofactors can make the determination of protein concentration highly inaccurate. Often it is necessary to consider not only proteins but also other reagents and buffers, such as reducing agents and detergents. The ideal method will be chemically compatible, reliable, inexpensive, and simple to set up.
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