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).

Offenlegungen

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

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Diesen Artikel zitieren
Liquid Phase Isoelectric Focusing: An Electrophoresis Technique to Separate Bioactive Molecules from Crude Gymnema sylvestre Extract. J. Vis. Exp. (Pending Publication), e21117, doi: (2023).

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