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Wednesday, July 4, 2012

Immobilized Metal ion Affinity Chromatography (IMAC) Principle & Applications

Immobilized Metal ion Affinity Chromatography (IMAC)- Principle & Applications

Affinity Chromatography Principle

Affinity chromatography is a technique used to separate biochemical molecules (eg: proteins) from a mixture (cell lysate or mixture of proteins) making use of the specific interaction of the molecule, interaction of antigen-antibody, interaction of protein to a specific ligand etc.

immobilized metal affinity chromatography

Affinity chromatography has got number of applications,
  1. Nucleic acid purification.
  2. Protein purification from cell free extracts.
  3. Purification of Proteins from Serum.
Immobilized Metal ion Affinity Chromatography (IMAC)

Immobilized metal ion affinity chromatography (IMAC) is based on the specific interaction of certain amino acids with the metals, especially histidine. Proteins which has affinity towards certain metals can be used for separating out target protein from a mixture of proteins. Nickel or Copper are used for purifying His-Tagged proteins and phosphorylated proteins are separated out using iron or zinc metal ions.

Chelators most widely used for IMAC are Nitrilotriacetic acid (NTA) or Iminodiacetic acid (IDA).

Ni-Affinity Chromatography is one such affinity chromatographic technique which utilizes the interaction of Nickel and Histidine for purifying the Histidine Tagged proteins (6X His Tag).

Polystidine tag is mostly used in E.coli expressed recombinant proteins, His tag will be present at the C or N terminal of the protein which is makes it easier in purification by affinity chromatography step. Mostly 6X His-Tag is used in recombinant proteins.

Nickel, Cobalt and Copper are most widely used metal ions for the purification of His-Tagged proteins.Nickel provides good binding efficiency to His-Tagged proteins but tend to have non-specific binding also. Cobalt provides more specific binding to histidine tags, cobalt based resin is used when protein is required in high purity. copper ions binds His-Tags more strongly but specificity is very low. purity of the separated protein can be checked using SDS-PAGE or Western Blotting.

Ni-Affinity Chromatography Principle and Uses

Ni-Affinity Chromatography uses the ability of Histidine (His) to bind to nickel. Six histadine amino acids (Polyhistidine tag / 6X His Tag) at the end of a protein (either N or C terminus) is known as a 6X His tag.

Nickel is bound to an agarose bead by chelation using nitrilotriacetic acid (NTA), NTA binds Ni2+ ions by four coordination sites.

Nickel ion chromatography
Qiagen Nickel ion chromatography: Image source: Qiagen

The general procedure involves batch binding of the protein, the protein mixture is mixed with the sample which allows the His-Tagged proteins to bind to Nickel of the chromatographic matrix, the slurry is then poured into a chromatographic column.

Lower concentrations of phosphate and imidazole are used to remove low affinity bound proteins, increasing the imidazole concentration results in the elution of all bound proteins from the column depending on their strength of binding.

Affinity chromatographic matrix / resin is commercially available, even prepacked columns are available (HisTrap HP from GE, Ni-NTA Agarose from Qiagen, etc)

Immobilized Metal ion Affinity Chromatography (IMAC) Methods

Sample Preparation:
Cells are harvested and lysed by mechanical or enzymatic methods, suitable lysing conditions are selected and need to be optimized for better results. In HisTrap™ FF from ge, cell lysate can be directly loaded onto the column.

Choice of Buffer:
Binding is done at near neutral pH.

Elution can be done using increased imidazole concentration. Suitable concentration of imidazole need to be used for optimum results.

Regeneration of Matrix:

The beads can be regenerated with 10 column volumes of the following:
  1. MES Buffer wash at pH 5.0,
  2. Wash with water,or20% EtOH.
Long term storage of Matrix / Resin can be done in 20% Ethanol (EtOH).

Thermo Scientific Technical Resources.
Ni-NTA Purification System, Life Technologies.
Bio-Techniques, International Journal of Life Science Methods.
Nickel Affinity Chromatography Protocol/Guide, DragonTech.