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

Overview

This video describes a technique to separate complex molecules from plant extracts using liquid phase isoelectric focusing. This technique helps purify complex biomolecules in their native state which can be used for various downstream applications.

Protocol

1. Separation and purification of small molecules and peptides from Gymnema sylvestre extract

1. Measure 0.6 g of plant extract and dissolve in distilled water (60 mL) by mixing in a roller tube for 5 min.
   NOTE: Any biological sample that is soluble and free of salt can be used for separation and purification using a liquid-phase Isoelectric Focusing (IEF) instrument. Samples with buffering salt concentration up to 10 mM can be used with slightly decreased resolution.
2. Centrifuge the solubilized plant extract at 10,000 x g for 5 min to remove insoluble particles.
3. Transfer the supernatant (~60 mL) to an 80 mL centrifuge tube and mix it with 0.6 mL of ampholyte (pH 3-10) to 1% (v/v).
4. Prepare the liquid-phase IEF unit.
   1. Assemble the liquid-phase IEF electrodes (anode-red button and cathode-black button) with their respective exchange membranes according to the instruction manual (Table of Materials). Equilibrate the anion exchange membranes with 0.1 M NaOH and the cation exchange membranes with 0.1 M H₃PO₄ at least for 16 h when new membranes are used.
   2. Store the membranes in electrolytes (0.1 M NaOH or 0.1 M H₃PO₄) between runs and do not allow to dry out.
   3. Assemble the inner and outer portion of the electrode by aligning three oblong holes in the ion-exchange gaskets. Fill the electrodes with respective electrolytes (~25-30 mL) to prevent their membranes from drying out.
   4. Do not add excess (more than 1/3rd of electrode chamber volume) electrolyte that may build up pressure inside the electrode and cause leakage.
   5. Cover the sample collection ports with sealing tape that comes with the instrument assembly parts (Table of Materials). The sample collection ports side can be identified by the two vertical metal aligning pins. Alternatively, use standard sealing tape to seal the ports.
   6. Assemble all parts of the focusing chamber assembly in sequence (anode electrode, nylon membrane core, focusing chamber and cathode electrode) over the ceramic cooling finger.
   7. Fill the focusing chamber with precooled distilled water (total volume of 60 mL for the standard IEF cell) using a 50 mL syringe.
   8. Connect the liquid-phase IEF instrument to a circulating cooling water at 4 ºC and prerun the unit at 15 W and 3,000 V for 3-5 min or until the voltage stabilizes.
      NOTE: Generally, within one minute, the voltage will reach the maximum set values. Prerunning with distilled water helps to remove the residual ions from the focusing chamber and the nylon membrane core.
   9. Switch off the power source and remove the water from the cell using the fraction collector. Re-seal the collection ports with sealing tape. The chamber is now ready to load the sample.
5. Use a 50 mL syringe with a 1-1/2 inch 19 G blunt end needle (comes with the instrument) and load the prepared sample with ampholyte (60 mL total) in the cell through sample collection ports.
6. Remove air bubbles from the sample cell by removing the focusing cell from the stand and tapping the electrode chamber to dislodge the bubbles. The presence of air bubbles will cause voltage and current fluctuations and affect the run.
7. Connect the unit to the water coolant (4 °C) and start fractionation with the power supply at a constant 15 W.
8. Run the apparatus for 3 h or until the voltage reaches a constant value.
   NOTE: As the sample starts focusing, the voltage will start climbing gradually until it reaches a constant value.
9. After the run, prepare to collect the fractions in the harvest box (containing 20 plastic tubes, 12 mm x 75 mm, 5-6 mL volume) connected to the vacuum pump by pressing the harvest button ON in the IEF unit.
10. Align the 20-collection pins with the 20-collection ports of the focusing cell that is sealed with the tape.
11. Push the collection pins through the sealing tape and turn the vacuum pump ON simultaneously (Figure 1B, 1C & 1D).
    NOTE: About 3 mL fractions from each chamber will be collected into the tubes for each time. Fractions can be used for subsequent downstream applications (SDS-PAGE for peptide quantification, TLC and bioassay for small molecules).

Representative Results

Figure 1:Liquid-phase IEF apparatus setup and fractionation of G. sylvestre plant extract. (A) During the run, (B) during fraction collection, (C) after fraction collection, and (D) liquid-phase IEF apparatus parts, 1) ion exchange membranes, 2) focusing chamber and membrane core, 3) electrode assembly (negative), 4) electrode assembly (positive).

Materials

Name	Company	Catalog Number	Comments
0.45 µm syringe filter	Fisher scientfic	09-720-004
Bio-Lyte 3/10 Ampholyte	Bio-Rad	163-1113
Bio-Lyte 5/8 Ampholyte	Bio-Rad	163-1192
Compact low temperature thermostat	Lauda -Brinkmann	RM 6T	Set water cooling to 4°C and it can be run even at 0°C as when it passes through the Rotofor cooling core, the circulating water temperature will be around 5 or more depending on the voltage.
Gymnema plant leaf extract powder (>25% Gymnemic acids)	Suan Farma, NJ USA
Mini protean electrophoresis	Bio-Rad
pH meter	Mettler Toledo	S20	Useful to determine the pH of the Rotofor (liquid-phase IEF) fractions
Rotofor	Bio-Rad	170-2972	http://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1702950E.pdf (Rotofor Instruction manual for assembling the unit)
RPMI-1640 Medium	HyClone	DH30255.01
Sealing tape	Bio-Rad	170-2960	Scotch tape may also be used
Sorvall legend micro 17 centrifuge	Thermo scientific	75002432