Imac chromatography: Difference between revisions
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IMAC - Immobilised metal affinity chromatography is one type of [[column chromatography|column chromatography]] used to seperate [[protein|protein]]/[[amino acids|amino acids]] from a mixture. This method of chromatography requires recombinant protein expression: the natural [[DNA|DNA]] sequence for a protein is taken and is engineered to include 6-10 histidine residues at the N or C terminus of a protein. This is nicknamed the 'His-Tag'. Genetic engineering is useed to produce a protein in a host such as bacteria. Histidine is a charged heteroaromati amino acid with a imidazole side chain which can bind to metals such as [[Nickel|Nickel]] or [[Zinc|Zinc]]. To seperate proteins not containing the His-Tag in a chromatography column, the mixture is ran down the column and all proteins containing a his-tag will elute out of the column. Once all other proteins have eluted, to then remove the desired proteins containing the His-Tag, you then add imidazole which is very similar to histidine. The [[imidazole|imidazole]] will compete with the histidine to bind with the Nickel or Zinc but has a higher affinitiy than the histidine so seperates it from the metal ions, allowing it to elute out of the column.<ref>http://www2.knauer.net/dwnld_fls/b_e_co_biofox_imac_tren_ida_17+40.pdf</ref><br> | |||
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Latest revision as of 22:56, 27 November 2014
IMAC - Immobilised metal affinity chromatography is one type of column chromatography used to seperate protein/amino acids from a mixture. This method of chromatography requires recombinant protein expression: the natural DNA sequence for a protein is taken and is engineered to include 6-10 histidine residues at the N or C terminus of a protein. This is nicknamed the 'His-Tag'. Genetic engineering is useed to produce a protein in a host such as bacteria. Histidine is a charged heteroaromati amino acid with a imidazole side chain which can bind to metals such as Nickel or Zinc. To seperate proteins not containing the His-Tag in a chromatography column, the mixture is ran down the column and all proteins containing a his-tag will elute out of the column. Once all other proteins have eluted, to then remove the desired proteins containing the His-Tag, you then add imidazole which is very similar to histidine. The imidazole will compete with the histidine to bind with the Nickel or Zinc but has a higher affinitiy than the histidine so seperates it from the metal ions, allowing it to elute out of the column.[1]