Proteins have different pH values due to their amino acids. The isoelectric point (pI) is when a protein has no net charge. Isoelectric focusing separates proteins with known pI from a mixture. Glycosylation can affect pI. Proteins can be sequenced and analyzed for purity.
Proteins are made up of chains of amino acids, each of which has different pH values. The overall pH of the protein is composed of the mixture of the pH values of the individual amino acids as they form ions in the particular solution in which they are dissolved. The isoelectric point (pI) of a protein is the pH at which that protein has no net charge. This property can be exploited to separate the protein with the known pI from other proteins in a heterogeneous mixture.
Amino acids have a terminal amino group which is basic, with a high pH. The other end of the amino acid is the carboxyl terminus which is acidic, with a low pH. At different pH values, the amino acids on proteins will vary in their charges. Proteins below their isoelectric point have a positive charge. Conversely, those above this point are negatively charged.
To exploit the knowledge of the isoelectric point for protein purification, a mixture of proteins is subjected to an electric field. This is commonly done in agarose or polyacrylamide gels and is known as isoelectric focusing. An older technique is to perform the procedure on a larger scale in a glass column using a sucrose solution with electrodes at each end. Compounds called ampholytes are added which cause a consistent pH gradient to form. When the gel or column is subjected to an electric current, the proteins migrate until they reach their isoelectric point, and then remain stationary.
The proteins on the gels are usually made visible by a protein-binding dye. Sometimes, if you’re studying enzymes, you can use a substrate that gives a color reaction. Standards that have proteins of known isoelectric points are usually used.
Once the location of the desired protein is known, a common technique is to cut the isolated protein from the gel. The protein can then be purified and sequenced. Once the sequence is known, it can be used to design polymerase chain reaction (pcr) primers and used to clone the gene for the protein if suitable nucleic acid material is available.
Isoelectric focusing is also a common way to analyze closely related proteins to see how different they are from each other. A complication can be that sugars may be bound to proteins. This is called glycosylation and can affect the pI of the protein. It may appear that there are multiple proteins with different isoelectric points, when in reality there is only one protein that has been differentially glycosylated. Proteins purified by standard methods such as chromatography are sometimes analyzed by isoelectric focusing to ensure their purity.
Protect your devices with Threat Protection by NordVPN
