What are the different methods of determining protein concentration?
There are hundreds of different methods of determining protein concentration. The incredible diversity in the types of protein solutions that biochemists analyze is why there is no only universal method that would work for each type of protein solution. The most common protein tests are Bradford test, Lowry test and bicinchoninic acid test. However, innumerable variations have been developed as needed to solve any potential chemical incompatibility between protein solution and agents used in the test.
Generally speaking, there are two main categories of determination of protein concentration. In the first group of methods, a color or fluorescent dye is added to the protein solution and is bound specifically to the protein. The bound dye has a unique absorption wavelength, which is proportional to the amount of protein. The use of the spectrometer can be estimated to estimate the protein concentration.
The Sedical Group of Tests Includes the addition of copper ions (II) to the protein solution where these ions RED areUsed for copper ions (i). These reduced ions are then able to form color complexes by binding to proteins. Measurement of absorbance on their unique wavelengths can also derive protein concentrations.
One of the most popular methods of determining protein concentration is Bradford test. In this test, a red dye called Coomassie brilliant blue is added to the protein solution under acidic conditions. Because this dye is bound to protein, it forms a permanent blue complex with characteristic absorbance on 595 nanometers.
Despite the general versatility of the Bradford test, it is incompatible with some protein solutions. In particular, the Bradford test is disrupted by the presence of sodium dodecylsulphate (SDS), a detergent that is commonly used to clean proteins and break down the cells. This detergent interferes with a dye binding to proteins, resulting in an unreliable and inaccurate absorbIt rises reading. Therefore, other types of methods should be used in the presence of SDS.
has developed another series of protein tests and all include a variant of the Biuret test. In this reaction, the protein is combined with an aqueous base and copper ions (II). These ions are reduced and then chewed with protein to form color complexes. The two tests that use this test are the Lowry test and the bicinchoninic acid test.
In the Lowry test, the folin-cocalteu agent is added to the biuret test. Folin-Cocalteu oxidizes aromatic residues, especially tryptophan, and helps the complex to absorb strongly at 750 nanometers. On the other hand, the bicinchoninic acid test includes the addition of bicinchoninic acid to the biuret test. After a short incubation at about 104 ° Fahrenheit (40 ° Celsius), two equivalents and peptide bonds of chelate protein (I) ion. The result is a complex that strongly absorbs on 562 nanometers.
When selecting a method for determining protein concentration is a minein order to consider the various chemical functional groups present in the solution. The presence of some side chains of amino acids, disulphide bonds and coffee -offs may cause the protein concentration to be wildly inaccurate. It is often necessary for a person to consider not only proteins but also other agents and buffers such as reduction agents and detergents. The ideal method will be chemically compatible and will be reliable, cheap and simple settings.