This article details the process of isoelectric focusing (IEF) using a pre-assembled IEF cell to separate components of a plant extract based on their isoelectric points (pI). The method involves applying a high voltage to create a pH gradient, allowing for the fractionation of differently charged components.
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.
Add 0.6 grams of Gymnema sylvestre plant extract to 60 milliliters of distilled water. Dissolve the plant extract by mixing in a roller tube for five minutes.
To remove the insoluble particles, centrifuge the solution at 10,000 x g for five minutes. Transfer the supernatant to a 150-milliliter glass beaker, and add ampholyte to create a 1% solution by volume, approximately 0.6 milliliters.
Using a 50-milliliter syringe, with a 1.5-inch, 19-gauge blunt-end needle, load the solution into the IEF cell through the sample collection ports. Then, remove the cell from the stand, and tap the electrode chamber to dislodge and remove any bubbles.
Connect the unit to the cooling water. With the power supplied at constant 15 watts, begin fractionation. Run the IEF unit until the voltage reaches a constant value, approximately three hours.
After pressing the Harvest ON button, align the fraction collector pins with the collection ports. Push the pins through the sealing tape to begin collecting the Gymnema sylvestre fractions.
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