Purifying and Separating Biomolecules

Why are IMAC and gel filtration combined?

IMAC (Immobilized Metal Affinity Chromatography) and gel filtration are often combined in separation processes to effectively purify and separate biomolecules based on their size and metal-binding properties.

IMAC is a technique that utilizes the specific interactions between metal ions (such as nickel, zinc, or copper) immobilized on a solid support and metal-binding groups on target proteins or biomolecules. It is commonly used for the purification of proteins with polyhistidine (His-tag), which can bind to metal ions with high affinity. By applying a sample containing the protein of interest to an IMAC column, the His-tagged protein will bind to the metal ions while other contaminants are removed in the process. Subsequently, the bound protein can be eluted by the addition of a metal chelating agent that competes for the metal ions, releasing the purified protein

However, IMAC alone may not provide sufficient purity or specificity, and this is where gel filtration comes into play. Gel filtration, also known as size-exclusion chromatography, is a technique that separates molecules based on their size and molecular weight. In this method, a stationary phase with porous beads is utilized, through which small molecules can enter the beads and take a longer path, while larger molecules pass through the beads more quickly, resulting in their separation

Gel filtration is often used after IMAC to further purify the His-tagged protein and remove any remaining contaminants or excess metal ions. By passing the eluted protein from the IMAC column through a gel filtration column, smaller molecules that might interfere with downstream applications are effectively separated from the target protein. This step can also be valuable for the removal of aggregates, as size-exclusion chromatography can distinguish between monomeric and higher-order assemblies

Combining IMAC and gel filtration in a purification process allows for a more comprehensive and effective purification strategy. IMAC provides selectivity based on the metal binding affinity of the target protein, while gel filtration helps to remove unwanted compounds and aggregate species, and ensure the final product is of high purity and suitable for further analysis or downstream applications

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