The Protein Man's Blog | A Discussion of Protein Research

Affinity chomatography is a routinely used technique for purifying or enriching a protein or molecule of interest through its specific binding affinity. The target protein adheres to a particular ligand that has been immobilized on a solid support (usually beaded agarose resin). This process produces high selectivity, resolution, and capacity for the protein of interest. Traditionally, affinity chromatography is performed in column format. Where the sample is applied and eluted by gravity flow through a packed resin bed of one to several milliliters. However, gravity flow columns are time consuming and require constant attention while supernatant filters through the column and resin. This process can be accelerated with G-Biosciences’ FastPure™ Spin Columns (Mini and Midi) specifically designed for simple and efficient small scale protein purifications. FastPure™ Spin Columns combine the effectiveness of gel filtration, and the speed of centrifugation for quick and reliable protein purification.

Chromatography is a versatile field with a wide range of applications. It’s accomplished by fractionating a mixture into its molecular components. Chromatography was first used in 1901 by Russian botanist Mikhail Tsvet when he realized the technique could separate plant pigments. It has since become widely developed and utilized for separation analysis in various scientific fields.

There are several contemporary methods for protein purification. Some techniques are very powerful, such as reverse phase chromatography, immunoadsorption, or affinity chromatography. However, these methods may ultimately denature protein and negatively impact the desired product. Likewise, recombinant proteins recovered from inclusion bodies are often aggregated and nonfunctional. In all of these situations, the purified protein product must be renatured for further use.

Salt precipitation can be a very powerful tool to purify proteins by precipitation. Ammonium sulfate is usually the salt of choice since it is cheap, very soluble in water, and is able to become much more hydrated (interacts with more water molecules) than almost any other ionic solvent. In practice, ammonium sulfate is either added directly as a solid or added as a (usually) saturated solution to precipitate desired proteins.