Affinity binding chromatography

From The School of Biomedical Sciences Wiki
Jump to: navigation, search

Affinity binding chromatography is the selection of molecules through chromatography by exploiting the high specficity of many protein and enzymes, it is therefore a very accurate way of selecting just one protein among many hundreds:

One method of affinity chromatography is the use of inhibitor molecules to select and purify a solution of enzyme, to do so the enzymes inhibitor must be placed in the chromatography column, as the solution is run through only the enzyme specfic to that inhibitor will bind and remain in the stationary phase, the rest of the molecules will be eluted. Then to capture the enzyme simply increase the concentration of free ligand also complimentary to the inhibitor in the solution to displace the enzyme.

Another method of affinity chromatography that does not rely on enzymes is immobalised metal affinity chromatography, this is common tool in the extraction of recombinant proteins from bacterial cells:

  1. Firstly modify the gene to be expressed in the bacteria to code for a series of 6-10 histidine residues at the end of the gene, this is called a His-tag, as this series of amino acids occure very rarely in nature and therefore will only exist on the protein you wish to select.
  2. Histidine has a high affinity for metals such as zinc and nickel and therefore will bind to them inside the elution column, run the mixture through several times to ensure purity.
  3. Elute the recombinant protein by returning it to the mobile phase through addition of Imidazole a chemical very similar to histidine that will displace it from the zinc and nickel.
  4. Collect the eluate recombinant protein.
Personal tools
Namespaces
Variants
Actions
Navigation
Toolbox