Explain how isoelectric focusing works.

• Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules based on isoelectric point differences.

• It is a type of zone electrophoresis performed on proteins in a gel that takes advantage of the fact that the overall charge on the molecule of interest is a function of its surroundings' pH.

• At isoelectric point, the average charge of all protein forms is 0 . Those proteins won'${}^{\mathbf{t}}$migrate in an electric field at their isoelectric${}^{\mathbf{pH}}$

• This is the basis of a protein separation technique ${}^{\mathbf{\to }}$isoelectric focusing.

• A protein solution is exposed to a strong electric field in a medium with a pH gradient:

• Positively charged molecules move to the negative pole

• Negatively charged molecules move to the positive pole

• Each of the proteins will migrate until they reach the point where the${}^{\mathbf{pH}}$ isoelectric pH.

• At an isoelectric point, the protein has no electric net charge and stops moving.

Therefore, each of the proteins in the solution is focused in one region at their isoelectric${}^{\mathbf{pH}}.$