IsoElectric Focusing (IEF)

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We have studied in previous lectures that proteins have ionizable amino acids side chains. Ionization state of these side chains and N-terminal amino group and C-terminal carboxyl provides net charge to a protein at given pH. As pH changes ionization state of the amino acids side chains also changes (can be described by the Henderson-Hasselbalch equation). This results in change in overall charge on protein.

Proteins with different amino acid sequences have different amino acid side chains composition. Thus, at a given pH net charge may be different. In other words, the charge on protein depends

At acidic pH most proteins are positively charged while in alkaline pH negatively charged. However, in between pH net charge on protein can vary considerably. This variation is because of different protein sequence or difference in post-translational modification. pH where net charge on a given protein is zero, called isolectric point of the protein. Isoelectric point is an important biophysical parameter.

If we do an electrophoresis in pH gradient (We shall also discuss how to make pH gradient during later part of the lecture) and protein sample (protein mixture) is loaded at pH 7.0: