Immobilized metal ion affinity chromatography IMAC

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]. 

The technique immobilized metal ion affinity chromatography (IMAC) is widely used to purify proteins (Experiment 4). Find two proteins that have recently been purified by this technique and briefly describe the methods for isolation. 

Next to RPLC, there are other LC modes that can be applied in protein separation, e.g., size-exclusion (SEC), ion-exchange (lEC), affinity (AfC), and immobilized metal-ion affinity chromatography (IMAC). However, in most cases high concentrations of nonvolatile salts have to be used to achieve the elution of proteins. All modes have been applied as the first separation step in two-dimensional (2D) LC systems for the separation of peptides (Ch. 17.5.4 and Ch. 18.3.2). 

Immobilized metal-ion affinity chromatography (IMAC) is a collective term that includes all kinds of affinity chromatography, where metal atoms or ions immobilized on polymer cause or dominate the interaction at the sorption site. 

Affinity separation of proteins in immobilized metal ion affinity chromatography (IMAC). The method is based on the fact that some protein molecules, having specific residues on the surface, can form complexes with metal ions immobilized on the sorbent matrix. Retention is determined by the coordination interactions forming the complexes. Immobilized metal ion affinity chromatography can be performed under very mild, nondenaturing conditions with extremely high selectivity it is particularly suitable for preparative group fractionation of complex extracts and bio-fluids. 

Protease-free preparation of CNTs can be obtained using an immobilized-metal-ion affinity chromatography (IMAC) step (Ros-setto et a/., 1992). This procedure is also useful for the purification of He, the 50 kDa carboxy-terminal part of the heavy chain of TeTx, which shows an identical retention time. However, IMAC-chromatography cannot be used for the purification of BoNT/D because this serotype is not retained. To obtain a protease-free BoNT/D preparation, an ion-exchange chromatography procedure was used (Schiavo and Montecucco, 1995). After freezing in liquid nitrogen, purified CNTs are stored at -80°C. 

Immobilized metal ion affinity chromatography (IMAC) rehes on the ability of certain amino acid side chains to form coordinate bonds with immobilized metal ion complexes [6,181-186]. The adsorption of proteins mainly takes place through interactions with the imidazole ring of histidines. For other amino acids with electron donor atoms in their side chains, binding tends to be weak. Cysteines in natural proteins, are rarely available in an appropriate reduced form for binding to chelated metal ions. 

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

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