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

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Samenvatting

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 decreased resolution.
Centrifuge the solubilized plant extract at 10,000 x g for 5 min to remove insoluble particles.
Transfer the supern....

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Resultaten

Rotofor Cell system for protein purification, electrophoresis chamber components diagram, Bio-Rad setup.
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,.......