Affinity chromatography is a method of separating protein mixtures, based on a highly specific biological interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. Affinity chromatography combines the size fractionation capability of gel permeation chromatography with the ability to design a stationary phase that reversibly binds to a known subset of proteins. The method was invented in 1968 (Cuatrecasas et al.) and Meir Wilchek for which the Wolf Prize in Medicine was awarded in 1987.
Possibly the most common use of affinity chromatography is for the purification of recombinant proteins. Proteins are tagged in order to aid their purification. The protein may have been genetically modified to allow it to be selected for affinity binding, this is known as a fusion protein. Tags include His-tags and GST (glutathione-S-transferase) tags. His6-tags have an affinity for nickel or cobalt ions which are coordinated with a chelator for the purposes of solid medium entrapment. For elution, an excess amount of a compound able to act as a metal ion ligand, such as imidazole, is used. GST has an affinity for glutathione. For elution, excess glutathione is used to displace the tagged protein. For review see Wilchek et al., 1999.
Service avaiable:
- All conventional columns available
- Special affinity columns upon individual request
Ion exchange chromatography (wikibooks) separates proteins according to the nature and degree of their ionic charge. The column to be used is selected according to its type and strength of charge. Anion exchange resins have a positive charge and are used to retain and separate negatively charged proteins, while cation exchange resins have a negative charge and are used to separate positively charged molecules.
The general procedure starts by buffering the column to equilibrate the opposing charged ions. Upon application of the sample, solute molecules (including proteins) will exchange with the buffer ions as each competes for the binding sites on the resin. The length of retention for each protein depends upon the strength of its charge. The most weakly charged compounds will elute first, followed by those with successively stronger charges. Because of the nature of the separating mechanism, pH, buffer type, buffer concentration, and temperature all play important roles in controlling the separation. For detailed overview see the handbook by GE/Amersham/Pharmacia.
Service available:
- Preparative pre-purification up to 5 grams of protein (Q- Sepharose Fast Flow)
- Analytic final purification (Mono-Q, Mono-S, Mono-P)
- Semi- preparative analytical purification (Mono-Q)
Gel filtration, also known as size exclusion chromatography (SEC), using cross-linked dextran as gel matrix was initially described by Porath et al. (1959). It is one of the simplest mechanisms for separating molecules. This method separates proteins based on their size. The stationary phase is packed with porous bead with a range of pore sizes. When a mixture of protein is introduced into the column, a range of protein from small, intermediate and large will be filtered through the column.
- Small proteins can fit inside any pores and enter many pores before reaching the end of the column. Thus the movement of small proteins are greatly impeded which cause them to elute last in SEC.
- Intermediate size protein may fit in some pores but not in all. These protein will generally elute somewhere between the small size and large size protein.
- Large size proteins cannot enter the beads. Their movements in the column are not impeded by beads which make them the first protein to be eluded from the column.
In practice, gel filtration can be used to separate proteins by molecular weight at any point in a purification scheme of a protein. It can also be used for buffer exchange.
Service available:
- Semi- preparative pre-purification up to 200 mg protein (Superdex 75, Superdex 200)
- Analytic purification (Superose 12, Superose 6, Poly-Sep, Bio-Sep)
Reverse Phase chromatography is a separation based on the solubility of the protein. This separation method for proteins was first described in the early 70s (Hofstee, 1972). The term ‘reverse’ was derived from its predecessor named ‘normal’ phase chromatography, which utilized a polar stationary phase such as silica. In reverse phase, the stationary phase is packed with silica modified with silyl ethers containing non-polar alkyl groups typically C4-C18 or biphenyl. This creates a hydrophobic stationary phase. A mobile phase of increasing hydrophobicity is used to dissociate the bound molecule at a point at which the hydrophobic interaction between the exposed patches and the immobilized matrix is less favorable than the interaction between the bound molecule and the solvent. The molecule releases from the matrix and elutes. For technical details review a handbook by Amersham/Pharmacia Biotech.
Service available:
- Analytic separation of protein solutions (Jupiter columns)
- Preparative pre-purification up to 5 grams of protein (Phenylsepharose, Butylsepharose)
- Analytic final purification (TSK- Phenyl)
Information partially taken from wikipedia figures are from Pharmacia publications.
