Question

Focusing techniques a) describe isoelectric focusing and isopycnic (density) separation techniques b) why are these real focusing techniques? c) how are these gradients established in both cases? d) describe the difficulties in using each e) why is chromatofocusing not a real focusing technique? f) describe advantages of these techniques and give examples Please provide reference

Answer 1

Isoelectric focusing

This is a real focusing technique because it is ideal for the separation of amphoteric substances such as proteins because it is based on the separation of molecules according to their different isoelectric points. This method has high resolution , being able to separate proteins by as little as 0.01 of pH unit. Separation is achieved by applying a potenital difference a gel that contains a pH gradient. The pH gradient is formed by the introduction of a gel of compounds known as ampholytes, which are complex mixtures of synthetic polyamino-polycarboxylic acids.

IEF is useful in studying microheterogeneity in a protein. For example, a protein may show a single band on an SDS-added gel, but may show three bands on an IEF gel. This may occur, for example, when a protein exists in mono-, di- and tri- phosphorylated forms. This method is also useful for separating isoenzymes and also be used for preparative purposes.

Isopycnic centrifugation:

There are two methods of density gradient centrifugation, the rate zonal technique and the isopycnic technique. The rate zonal technique is based upon the differences in shape, size and density of the particles, the density and viscosity of the medium and the applied centrifugal field. Isopycnic centrifugation depends solely upon the buoyant density of the particle and not its shape or size and is independent of time. The technique is used to separate particles of similar size but of differing density. Hence, soluble proteins which have a very similar density cannot usually be separated whereas subcellular organelles can be effectively separated. The method involves the layering of the sample on top of a gradient that spans the whole range of the particle densities that are to be separated. The maximum density of the gradient, must always exceed the density of the most dense particle. The particles band to form zones each at their own characteristic buoyant density.

This method is used to separate and purify viruses and analyse the human plasma lipoproteins, to study the variation of buoyant density wiht pH for proteins and homopolypeptides and has found wide application in the analysis of nucleic acids.

Chromatofocusing:

Its principle is same as isoelectric focusing, a linear pH gradient is generated in a column by exploiting the high buffer capacity of an ion-exchanger pre-equiliberated to a particular pH. This method is not a real focusing technique because it gives a good resolution of quite complex mixtures of proteins, provided that there are discrete differences in their isoelectric point. Proteins possessing very similar isoelectric points tend to be poorly resolved