In Biochemistry, researchers believe that you should never waste your pure thoughts thinking about impure proteins. Beginning with pure proteins, scientists can establish the amino acid sequence and the evolutionary relationships that exist between proteins in different organisms. In addition, one can also determine the biochemical function of a specific protein. Pure proteins can be used to grow crystals of proteins that can be used to mine x-ray data that will provide the tertiary structure of the protein, which is the actual functional unit. This is why protein purification is an essential technique in scientific research. Isoelectric focusing is one of the most important techniques used in purification of proteins. Read on to know more about this essential biochemical method.
Isoelectric focusing is a great analytical tool that is used in the separation of proteins and other types of amphoteric molecules for checking purity and determining their isoelectric points (pl). IEF can also be used in analyzing protein microheterogeneity. Isoelectric point standards are necessary to guarantee high performance of the analysis. Isoelectric focusing uses electrical charge properties of the molecules to center them in the well-defined zones of the separation medium. This focusing mechanism is what separates IEF from other separation techniques and makes it distinct from most separation methods.
Protein purification involves a series of different processes to isolate a specific protein from a complicated mixture. Isoelectric focusing is crucial for characterizing the structure, interaction, and function of the specific protein that one is interested in. An analytic purification mainly uses three different properties for the separation of proteins including the isoelectric method.
A distinct protein can be purified depending on its protein isoelectric point by running it through a pH-graded gel or an ion exchange column. A protein does not have a net charge at its isoelectric point. A protein will also carry a net positive charge below its isoelectric point and a negative charge above its pl point. Mybiosource.com has all the information you need about IEF techniques.
Since most of the weakly acid remains in almost every protein, they tend to be negatively charged at neutral pH. The Isoelectric point becomes significant in the purification process of proteins because it is a representation of pH where the solubility is classically minimal. The isoelectric point of the protein signifies the point at which the mobility is zero in an electro-focusing system and the point where the protein will accumulate.
In order to purify proteins using the isoelectric focusing technique, you need to mix a specific polymer with a uniform amount of protein solution. The polymer used has unique properties that ascertain that the proteins create a pH gradient when an electric field is introduced across the solution. After the development of this pH gradient, the protein molecules will simultaneously migrate in the up until the molecules get to their isoelectric point. Therefore, this powerful method purifies complex protein mixtures by assisting the proteins to achieve their individual isoelectric points.
Isoelectric focusing is a reliable technique that has been used for many years to purify proteins. This method has developed immensely over the years and is preferred because of its accuracy. IEF offers a more effective alternative to the customary electrophoresis used for genetic marker typing. IEF resolves allelic products depending on their electric points instead of their charge-to-mass ratio. It is a technique that scientists will continue to use for many years.