For example, a glutathione S-transferase (GST)-tagged fusion protein can be first captured to a glutathione support via the glutathione-GST affinity interaction and then secondarily chemically crosslinked to immobilize it. However, indirect coupling approaches are also possible. Most commonly, ligands are immobilized or “coupled” directly to solid support material by formation of covalent chemical bonds between particular functional groups on the ligand (e.g., primary amines, sulfhydryls, carboxylic acids, aldehydes) and reactive groups on the support (see related article on Covalent Immobilization). The target protein is eluted in a purified and concentrated form.Ī single pass of a sample (cell lysate, cell culture supernatant, or serum) through an affinity column can achieve greater than 1000-fold purification of a specific protein so that only a single band is detected after gel electrophoresis (e.g., SDS-PAGE) analysis. Elution conditions may be specific, such as a competitive ligand, or nonspecific, such as changing pH, ionic strength, or polarity.
The bound protein is then recovered in a highly purified form by changing conditions to favor elution. The protein of interest is tightly bound to the resin under conditions that favor specific binding to the ligand, and unbound contaminants are washed off. Affinity chromatography is very selective and provides high resolution with an intermediate to high sample loading capacity. Small scale affinity purification using an antibody immobilized to a solid support. Chromatography has three main components: the mobile phase or solvent containing proteins, the stationary or solid phase also called the medium or resin (which may be agarose or other porous resin) and the chromatography column.
After other sample components are washed away, the bound molecule is stripped from the support, resulting in its purification from the original sample.Įach specific affinity system requires its own set of conditions and presents its own peculiar challenges for a given research purpose. A particular ligand is chemically immobilized or “coupled” to a solid support so that when a complex mixture is passed over the column, those molecules having specific binding affinity to the ligand become bound. In ion exchange chromatography, molecules are separated according to the strength of their overall ionic interaction with a solid phase material (i.e., nonspecific interactions).īy contrast, affinity chromatography (also called affinity purification) makes use of specific binding interactions between molecules. Gel filtration (also called size-exclusion chromatography or SEC) uses a porous resin material to separate molecules based on size (i.e., physical exclusion). Most purification methods, however, involve some form of chromatography whereby molecules in solution (mobile phase) are separated based on differences in chemical or physical interaction with a stationary material (solid phase). Selective precipitation is perhaps the simplest method for separating one type of macromolecule from another. Proteins and other macromolecules of interest can be purified from crude extracts or other complex mixtures by a variety of methods.