Immobilised metal-affinity

Phosphoproteins (various). Purified by adsorbing onto an iminodiacetic acid substituted agarose column to which was bound ferric ions. This chelate complex acted as a selective immobilised metal affinity adsorbent for phosphoproteins. [Muszyfiska et al. Biochemistry 25 6850 1986.]... 

Figure 2.26 Immobilised Metal Affinity Chromatography (IMAC). Metal ions (for instance, Ni + ions) are gel-immobilized by chelation to groups covalently attached to the solid support. Histidine (His) residues in proteins (or part of a His-tag) have a high affinity for the immobilized ion. Elution is typically carried out by using a competitively high concentration of imidazole.

Gels maybe eliminated by shotgun proteomics, but specihc protein modihcations of interest need to reviewed and resolved by some form of chromatography prior to mass spectrometry. For example, phosphopeptides maybe specihcally purihed pre mass spectrometry using immobilised-metal affinity chromatography (IMAC) (see Chapter 2). Both iron (as Fe +) and... 

Clemmitt R.H. and Chase H.A. 2000. Immobilised metal affinity chromatography of beta-galactosidase from unclarified Escherichia coli homogenates using expanded bed adsorption, J. Chromatogr. A, 874, 27. 

Another very important form of affinity chromatography is immobilised metal ion affinity chromatography (IMAC) (Kagedal 1989 Yip and Hutchens 1996). This... 

Willoughby N.A., Kirschner T., Smith M.P., Hjorth R. and Titchener-Hooker N.J. 1999. Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker s yeast using an expanded bed adsorption system, J. Chromatogr. A, 40, 195-204. 

Chaouk H. and Hearn M.T. 1999. New ligand, N-2-pyridylmethyl aminoacetate, for use in the immobilised metal ion affinity chromatographic separation of proteins, J. Chromatogr. A, 852, 105. 

Cu(II) has been shown to be bound by two cysteines [32]. The main limitation of the use of cysteine or glutathione for detecting metal ions is the thiol side chain which having a high affinity for soft metals is being employed to immobilise the peptide and hence is not available for coordination with the metal ion. 

Pirkle or brush type bonded phases Helical chiral polymers (polysaccharides) Cyclodextrins and crown ethers Immobilised enzymes Amino acid metal complexes Three-point interaction Attractive hydrophobic bonding Host guest interaction within chiral cavity Chiral affinity Diastereomeric complexation... 

Example of the variety of tails which have been used are shown in Table 9-4. Some examples of the use of affinity tails demonstrates the versatility of the technique. Mouse dihydrofolate reductase with a polyhistidine peptide tail was purified by using metal chelate chromatography followed by removal of the affinity peptide with car-boxypeptidase A [38], A short hydrophilic antigenic peptide of eight residues engineered on to the N-terminus of recombinant lymphokines allows them to be purified by using an immobilised monoclonal antibody specific for the first four amino acids of the peptide [39]. 

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