Liquid Phase Isoelectric Focusing: An Electrophoresis Technique to Separate Bioactive Molecules from Crude Gymnema sylvestre Extract
Published: April 30, 2023
Abstract
Source: Veerapandian, R., et al. Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method. J. Vis. Exp. (2020).
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 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 decrea…
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).
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Cite This Article
Liquid Phase Isoelectric Focusing: An Electrophoresis Technique to Separate Bioactive Molecules from Crude Gymnema sylvestre Extract. J. Vis. Exp. (Pending Publication), e21117, doi: (2023).