Isoelectric focusing electrophoresis- Principle , procedure and applications
- 1. ISOELECTRIC FOCUSING ELECTROPHORESIS ASSIGNMENT BY – JASKIRAN KAUR
- 2. PRINCIPLE • Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). 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. • The resolution of Isoelectric Focusing is very high. In normal electrophoretic methods pH between anode and cathode remains constant but in Isoelectric focusing , a pH gradient is arranged. When pH of protein is below its pI proteins become positively charged and it will migrate towards cathode. • Because of the pH gradient charge of the protein molecule changes while moving forward, there will be a point at which net charge of the protein becomes zero this point is called Isoelectric point. when a protein mixture or a single protein is run under an electric field at specific pH the protein stops moving.
- 4. EQUIPMENTS • In most cases, a commercially available immobilized pH gradient (IPG) strip is used. • The IPG strip consists of an acrylamide gel that contains wide pores to prevent a sieving effect based on protein mass, with a pH gradient. • Various gradients are available, with wider gradients, such as pH 3-10 that are used for whole proteome analysis, and narrower ranges, such as pH 5-8 that are used for more specialist applications. • The sample is usually combined with carrier ampholytes to assist in migration. Ampholytes are a mixture of charged molecules with a range of pIs that matches the pI range of the IPG strip. • The migration of the ampholytes encourages the sample molecules to move along the pH gradient. • Ampholyte mixtures of a variety of pI ranges are commercially available. After separation across the pH gradient, the sample is further separated (in 2D-PAGE) or analyzed
- 5. PROCEDURE • Setting up an IEF experiment is probably easier than understanding the theory behind what’s actually happening: 1. The IPG strips are rehydrated (face down) in a denaturing buffer (>6 M urea) with detergent, usually including carrier ampholytes (it’s a case of trial and error, but for many samples, it works better to add the ampholytes with the sample). 2. There are 2 ways to actually load the sample–you either include it in the rehydration buffer or you load it in a small plastic cup at the end of strip (again, some samples work better with one or other way of loading–give both a try and see what works best for you). 3. The strips are then placed in the IEF apparatus, filter paper wicks are placed over the ends of the gel (these collect salts and proteins/peptides that are outside of the pI range of the IEF strip) 4. Electrodes are placed on top
- 6. GEL slab used for IEF
- 8. APPLICATIONS OF ISOELECTRIC FOCUSING • In forensic serology :The typing of certain polymorphic proteins present in human body fluids is an important aspect of the analysis of serological evidence. This is particularly true when dealing with evidence related to violent criminal activity such as homicide, assault, or rape. • For separating , characterizing and purifying proteins and peptides. • Used in limit test when the density of band is compared with • the density of band of std prep. • For research in Taxonomy , Cytology and Immunology etc. • IEF gel is used as identity test when migration pattern on gel is compared with std preparation. • Isoelectric focusing (IEF) offers an effective alternative to conventional electrophoresis for genetic marker typing.
- 9. THANK YOU