This method is used to separate protein according to their iso-electric points. A stable pH gradient is established in the gel by the addition of ampholytes. A protein mixture is placed in a well on the gel. With an applied electric field, protein enters the gel and migrates until each reaches a pH equivalent to its pI. Iso-electric focusing and SDS electrophoresis are combined sequentially in a process called two-dimensional electrophoresis. It is a more sensitive method than either of the above mentioned electrophoresis; it separates protein of identical molecular weight differing in Pi or vice-versa.
Such as an example of mixtures which is used as gel are called Ampholine and Pharmalyte have mixtures of 600 to 700 different homologues of amphoteric compounds with a spectrum of isoelectric points between 3 and 10 form a pH gradient under the influence of the electric field. Their buffering capacities is very high at their isoelectric points. Their molecular weights below 1 kDalton and these are hydrophilic.
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