Write short note on isoelectric focusing.
Answers
Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI).[1][2] It is a type of zone electrophoresis, usually performed on proteins in a gel, that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.
This technique was discovered by H.Svensson in Sweden and has a high resolution power. Isoelectric focussing resolves plasma proteins into at least 60 bands. protein molecules have a net positive charge in an acidic solution because most amino groups carry a net positive charge and most carboxylic groups are protonated and electrically uncharged. with this gradual increse in ph ,the no. of carboxyl group carrying a negative charge increases while no. of positively charged groups decreases. At a certain ph value, the isoionic point, the net charge of the protein molecule is zero. The isoelectronic point of molecule is thus determined by the no. and types of protolytic groups and their dissociation constants. Although there is considerable variation in the isoionic point of proteins ,they are generally in pH range of 3-11.in isoelectric focussing also a stable pH gradient is arranged.the pH increases gradually from anode to cathode. Due to presence of pH gradient ,the protein migrates to an environment of higher pH value which influences the ionisation and net charge of the molecule . Protein encounters a ph where it is zero and stop migrating.this is the isoelectric point of protein.