Iso-electric focussing

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Iso-electric focussing (IEF) is a molecular separation technique, often used to separate proteins. 

IEF works on the basic principle of a proteins isoelectric point (pI). This is the pH at which a protein has an overall charge of zero.


Firstly a gel is established with a pH gradient. This is acheived by the electrophoresis of a mixture of polyampholytes (small multi-charged polymers).

You then add the sample in the middle of the gel and apply a voltage. This will involve a cathode at one end and an anode at the opposite end of the gel.

The proteins will then start to move according to their charge. Positively charged proteins will move towards the cathode, and negatively charged proteins will move towards the anode. Once the proteins reach a point on the gel where the pH = pI, the protein will stop moving.

This separation technique is so precise, it can separate proteins with as little as one net charge differences [1]


When the proteins reach their pI, they form bands, these can be removed and used in further experiments.


  1. Berg J., Tymoczko J., Stryer L., (2012) Biochemistry, 7th Edition, New York: W. H. Freeman and Company. pg 75.
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