What are the commonly used chromatographic methods for protein purification?

Commonly used chromatographic methods for protein purification include:

Ion exchange chromatography (IEXC) – This is a common protein purification method that separates different types of proteins based on their net charge. In this process, columns can be prepared to facilitate either cation exchanges or anion exchanges. While passing through the column, soluble molecules bind to the oppositely charged insoluble stationary phase.  Advantages of ion exchange chromatography are its high protein binding capacity, effectiveness in removing undesirable impurities, and the relative ease with which it can be performed.

Affinity chromatography – This protein purification technique separates biochemical mixtures based on highly specific interactions between different types of proteins and particular ligands. In this process, the protein of interest binds to a particular ligand under favorable conditions and is separated from the other undesirable unbound molecules that are washed away. Affinity chromatography is used to purify target proteins with well-defined properties.

Column chromatography - One of the most common methods of protein purification, column chromatography is based on the principle of selective adsorption. In this process, the chemical mixture is passed through a column of an adsorbent material. The molecules in the mixture migrate at different rates depending, hampering the movement of some molecules. The target molecules finally flow through the column after eluting with a select buffer.

Gel filtration or size-exclusion chromatography - This chromatography technique separates molecules based on their size and molecular weight. It exhibits good sensitivity and is generally used to separate larger proteins from smaller ones by using a minimal volume of eluate. There’s no sample loss in this process because of the absence of interaction between the solutes and the stationary phase.

Hydrophobic interaction chromatography – In this chromatography technique, the column containing the sample is treated with a highly ionic buffer. Target proteins with hydrophobic amino acid side chains on surfaces interact with the hydrophobic group. The bound proteins are then extracted.