Metal-chelate affinity chromatography

Affinity chromatography Ion-exchange chromatography (Metal-)chelate chromatography Size-exclusion chromatography Normal/reversed-phase chromatography... 

Immobilized Metal Ion Affinity Chromatography (IMAC) Chelating Sorbents... 

Ligand exchange Equihbrium Chromatographic separation of glucose-fructose mixtures with Ca-form resins Removal of hea y metals with chelating resins Affinity chromatography... 

Porath, J., Carlsson, J., Olsson, I., and Belfrage, G., Metal chelate affinity chromatography, a new approach to protein fractionation, Nature, 258, 598, 1975. 

Patwardhan, A.V. and Ataai, M.M., Site accessibility and the pH dependence of the saturation capacity of a highly cross-linked matrix. Immobilized metal affinity chromatography of bovine serum albumin on Chelating Superose, /. Chromatogr. A, 767, 11, 1997. 

Metal-chelate affinity chromatography is a powerful purification technique whereby proteins or other molecules can be separated based upon their ability to form coordination complexes with immobilized metal ions (Porath et al., 1975 Lonnerdal and Keen, 1982 Porath and Belew, 1983 Porath and Olin, 1983 Sulkowski, 1985 Kagedal, 1989). The metal ions are stabilized on a matrix through the use of chelating compounds which usually have multivalent points of interaction with the metal atoms. To form useful affinity supports, these metal ion complexes must have some free or weakly associated and exchangeable coordination sites. These exchangeable sites then can form complexes with coordination sites on proteins or other molecules. Substances that are able to interact with the immobilized metals will bind and be retained on... 

One limitation to this method should be noted. If the antibody-enzyme conjugate is prepared using antibody fragments such as Fab or F(ab )2, then nickel-chelate affinity chromatography will not work, since the requisite Fc portion of the antibody necessary for complexing with the metal is not present. 

Lonnerdal, B., and Keen, C.L. (1982) Metal chelate affinity chromatography of proteins. J. Appl. Biochem. 4, 203-208. 

Figure 6.17 Schematic representation of the basic principles of metal chelate affinity chromatography. Certain proteins are retained on the column via the formation of coordinate bonds with the immobilized metal ion (a). The actual structure of the most commonly used metal chelator, iminodiacetic acid, is presented in (b)...

Metal chelate affinity chromatography finds most prominent application in the affinity purification of recombinant proteins to which a histidine tag has been attached (described later). As protein binding occurs via the histidine residues, this technique is no more inherently useful for the purification of metalloproteins than for the purification of non-metalloproteins (a common misconception, given its name). 

Metal-chelate affinity chromatography (Immobilized-metal (Ion) affinity chromatography)... 

In 1970s, first application of metal-chelate affinity chromatography which is later named as "immobilized-metal (ion) affinity chromatography (IMAC) was perfomed. Metal-chelate chromatography technique exploits selective interactions and affinity between transition metal immobilized on a solid support (resin) via a metal chelator and amino acid residues which act as electron donors in the protein of interest [25-26]. As well as aromatic and heterocyclic compounds, proteins such as histidine, tyrosine, tyriptophane and phenylalanine posses affinity to transition metals which form complexes with compounds rich in electrons [25,27]. 

Fanou-Ayi L, Vijayalakshmi M. Metal-chelate affinity chromatography as a sepera-tion tool. Biochemical Engineering III 1983 413 300-306. 

Both BFD variants L476Q and 55E4 were overexpressed in E. coli BL21 (DE3) as His-tag fusion proteins and purified to electrophoretic homogeneity as detected by SDS-PAGE by metal chelate affinity chromatography using Ni-NTA (data not shown). 

Elimination of coextracted materials and concentration of tetracyclines have also been accomplished using mixed-phase extraction membranes with both re-versed-phase and cation-exchange properties (294,295), or solid-phase extraction columns packed with cation-exchange materials such as CM-Sephadex C-25 (301), aromatic sulfonic acid (310), and carboxylic acid (283, 300). For the same purpose, metal chelate affinity chromatography has also been employed. In this technique, the tetracyclines are specifically absorbed on the column sorbent by chelation with copper ions bound to small chelating Sepharose fast flow column (278-281, 294-296). 

Ultrafiltration (278, 279) and immunoaffinity chromatography (282) have also been described for removal of matrix components from milk extracts, while online trace enrichment has been reported for isolation/purification of tetracycline, oxytetracycline, demeclocycline, and chlortetracycline residues from animal tissues and egg constituents (305). The latter technique involves trapping of the analytes onto a metal chelate affinity preconcentration column (Anagel-TSK Chelate-5PW), rinsing of coextracted materials to waste, and finally flushing of the concentrated analytes onto the analytical column. 

In a different approach, Cooper et al. (305) developed an online metal chelate affinity chromatography-liquid chromatographic method for the determination of oxytetracycline, tetracycline, demeclocycline, and chlortetracycline residues in animal tissues and egg. According to this method, a 2 g blended egg or thinly sliced tissue is homogenized with citrate buffer pH 5 (pH 4, for chicken... 

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