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