Ion Exchange Chromatography for GNA Lectin Purification

2023-10-24

Ion Exchange Chromatography for GNA Lectin Purification

Ion exchange chromatography is a widely used technique for separating ionizable molecules, especially proteins and enzymes, based on their charge. For the purification of GNA lectins, ion exchange chromatography using an anion exchanger is employed. GNA lectins are stable over a wide pH range (3-12), making this method ideal for separating them from other proteins in a bulb extract. 

Since the pI (isoelectric point) of GNA lectins is approximately 3.5, an anion exchanger column is equilibrated with a 20 mM 1,3-diaminopropane buffer (pH 11). This creates an environment where the pH is higher than the pI of GNA lectins (pH > pI), deprotonating them and rendering them negatively charged.

During the loading step, proteins with a pI higher than the surrounding pH remain positively charged and are not retained by the column, while GNA lectins bind to the positively charged anion exchangers. 

To elute the GNA lectins, a salt-containing buffer (20 mM 1,3-diaminopropane at pH 11 with 0.5 M NaCl) is added. The salt ions displace the bound proteins due to their higher charge density, leading to the release of GNA lectins from the column.

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