05-02-2013, 02:27 PM
Isoelectric Focusing
Isoelectric Focusing.pptx (Size: 486.66 KB / Downloads: 23)
Introduction
Isoelectric focusing is a technique for seperating different biomolecules on the basis of their isoelectric points in a stabilized pH gradient.
Only applicable for amphoteric molecules.
Harry Rilbe and Tiselius were the major scientists to work on isoelectric focusing.
Principle
Protein in a mixture can be precipitated depending on its isoeletric point.
IEF requires stable pH gradient which can be formed by using mixture of specially designed amphoteric molecules known as ampholytes.
When electric field is applied, a pH gradient is established, that is negatively charged ampholytes moves towards anode and positively charged towards cathode and align themselves according to their pIs.
Sample loading
a) by first soaking it in filter paper and then to gel by applying voltage.
b) by directly introducing sample the column.
Finally voltage is applied and migration of molecules start.