What is the isoelectric point?

Proteins

are built of amino acid chains, each with different pH values. The total pH of the protein is composed of a mixture of pH values ​​of individual amino acids because they create ions in a particular solution in which they are dissolved. The isoelectric point (PI) of the protein is a pH in which this protein does not have a clean charge. This feature can be used to separate protein with known pi from other proteins in heterogeneous mixture. Amino acids

have an amino terminal group that is basic and has a high pH. The second end of the amino acid is a carboxyl terminal that is acidic, with a low pH. At different pH values, amino acids on proteins will differ in their charge. Proteins below their isoelectric point have a positive charge. On the other hand, they have a negative charge over this point.

If you want to use the knowledge of the isoelectric point for rotteine ​​picching, the mixture of proteins is subjected to an electric field. ThatIt commonly performed in agarosis or polyacrylad gels and is known as an isoelectric focus. The older technique is to perform a larger scale procedure in a glass column by means of sucrose solution with electrodes at each end. Compounds called ampolytes that cause the formation of a consistent pH gradient. When a gel or column is exposed to an electric current, the proteins migrate until they reach their isoelectric point, and then remain stationary.

Proteins on gels are usually visible by dye that binds proteins. Sometimes, if enzymes are studied, a substrate that gives a color reaction can be used. Usually standards that have proteins have known isoelectric points are used.

As soon as one knows where the required protein is placed, the common technique is to cut the isolated protein from the gel. Protein can then be purified and sequenched. Once the sequence is known, it can be used to design primers for a polymerase chain reaction (PCR) and afterUsed to clon the protein gene if suitable nucleic acid material is available.

isoelectric focusing is also a common way to analyze narrowly related proteins to see how they differ. One complication may be that proteins can have sugars on them. This is called glycosylation and can affect the protein. It may seem that there are more proteins with different isoelectric points, where there is actually only one protein that has been differently glycosylated. Proteins purified by standard methods such as chromatography are sometimes analyzed by isoelectric focus to ensure their purity.