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JoVE Encyclopedia of Experiments
Encyclopedia of Experiments: Biological Techniques

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Liquid Phase Isoelectric Focusing: An Electrophoresis Technique to Separate Bioactive Molecules from Crude Gymnema sylvestre Extract

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Begin with a pre-assembled isoelectric focusing, IEF cell - a liquid-phase electrophoresis unit. This column is a compartmentalized, horizontal porous chamber with distinct positive and negative terminals at the ends.

Now, take the plant extract containing a complex mixture of differently charged components with varying isoelectric points, pI - a pH where the net charge on a molecule is zero.

The extract is supplemented with ampholytes of variable charges that facilitate the fractionation of sample components later.

Load the sample into the loading ports of the column. Currently, the IEF column has a constant pH. Next, apply high voltage to initiate the electrophoresis.

Under the electric field, the soluble ampholytes migrate toward oppositely charged electrodes and establish a linear pH gradient across the column. Sample components start migrating through the liquid phase pH gradient, and ones positioned at pH below their pI carry a net positive charge.

These migrate toward the negative electrode until the components reach their pI, where their charge neutralizes, and migration stops. Similarly, components at pH above their pI migrate toward the positive electrode and stop when they reach their pI. This separates sample components based on their pI into focused compartments. Harvest the purified fractions for further analysis.

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