Immobilized metal affinity column

Figure 16.25 Experimental breakthrough curves of myoglobin on an immobilized metal affinity column (dotted line) and model calculations as a dimerizing system (solid line). Reproduced with permission from R. D. Whitley, K. E. van Cott, J. A. Berninger, N.-H. L. Wang, AIChE J., 37 (1991) 555 (Fig. 2).

Many of the techniques used to enrich PTMs at the protein level are applicable at the peptide level. A popular method for enrichment of phosphopeptides is to use an immobilized metal affinity column. The molecular basis for the enrichment is the phosphate affinity to transition metal ions, such as copper, nickel, cobalt, iron, aluminum, gallium, and zinc (142). A comprehensive source for phosphopeptide enrichment strategies can be found in a recent review (143). Lectin-based chromatography is used to enrich for glycopep-tides (144). Lectins are proteins that have an affinity for carbohydrates. With multiple available enrichment options, choosing the right one for the experiment is important. For determining which proteins have a certain PTM, protein-level enrichment can be performed. However, if a type of PTM is of... 

Metal affinity columns can be used for the purification of antibodies with a hexa-Histidine tag. Immobilized metal affinity chromatography is incompatible, in our own experience, with direct loading of antibodies in supernatants... 

Displacement of moderately retained proteins (e.g., lactoferrin and RNase A) has been carried out in immobilized metal affinity (IMAC) stationary phases using myoglobin as the displacer,55,56 While, imidazole was employed as the mobile phase modifier in these studies it was subsequently established that it possessed sufficient affinity to act as a displacer for proteins in IMAC systems.57,58 Figure 5 shows the separation of RnAse and myoglobin using imidazole as a displacer on a Cu2+ charged Sepharose column. To date,... 

Immobilized metal affinity chromatography has been shown to be effective for isolating proteins from crude mixtures, as well as for selective separations of closely related proteins [2]. With respect to separation efficiency, IMAC compares well with biospecific affinity chromatography and the immobilized metalion complexes are much more robust than antibodies or enzymes. These factors make IMAC particularly well suited for scale-up to process scale chromatography. The main scale-up points to be aware of are the degree to which the column is metal saturated, the chelating agent content of the sample, and the potential of leached metal (or its interactions) within the product eluate. 

Immobilized Metal Affinity Chromatography. The buffers used in the initial His-tag purification are detailed in Table II. The 50 mL lysate is loaded onto a 1 mL NP -charged HiTrap Chelating HP column (Amersham Pharmacia, Cat. 17-0408-01) nsing the AKTA FPLC system with Frac-950 fraction collector (Amersham Pharmacia). The pnrification scheme is detailed in Table III. The His-tagged Sec23/24 complex should elute at an Imidizole concentration aronnd 150 mM with a typical chromatogram and resultant western blot of fractions shown in Fig. 2. 

However, HPLC techniques exist that do utilize affinity chromatogaphy properties. Immobilized Metal Affinity Chromatography (IMAC) is useful to separate aforementioned molecules based on the relative affinity for the metal (I.e. Dionex IMAC). Often these columns can be loaded with different metals to create a column with a targeted affinity. 

A higher value application, based on functionalization of a solid particle, is the development of the stationary phase of nanoengineered analytical immobilized metal affinity chromatography columns by ATRP for separation of proteins and synthetic prion peptides. 

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)...

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