Imidazole affinity chromatography

Figure 3 Biosynthesis and purification of 90-kD elastin analogue analyzed by denaturing polyacrylamide gel electrophoresis (10-15% gradient, visualized by silver staining). Lanes 1-7 time course of target protein expression at 0, 30, 60, 90, 120, 150, and 180 minutes after induction. Lane 9 soluble lysate of induced E. coli expression strain BLR(DE3)pRAMl. Lanes 10-13 protein fractions obtained from immobilized metal affinity chromatography of the lysate on nickel-NTA agarose (imidazole gradient elution). Lanes 8,14 protein molecular weight standards of 50, 75, 100, and 150 kD.

As is common to all affinity chromatography protocols, only the desired protein is supposed to bind to the column. The target protein then has to be eluted off the column, in this case with 0.5 m imidazole which displaces the imidazole rings of the histidine residues of the target protein. Non-specific binding is often observed, which results in several intermittent washing steps being required as well as optimization of the protocol for each protein. 

Elution is carried out by reducing the pH and increasing the ionic strength of the buffer or by adding EDTA to the buffer. The most typical method is to gradually add sodium chloride (0.5-1.0 M) or imidazole (0-0.05 M). This ligand is very expensive, so metal chelate affinity chromatography is used only for small scale purification. 

Affinity Separation - Treatment of dCTP with carbonylbis(imidazole) followed by N-trifluoroacetyl-6-aminohexyl phosphate affords (191), which was deacetylated and coupled via its aminoalkyl group to CNBr-Sepharose, affording a P4 (dC) affinity column (192).326 This column gave a one-step, 19,000-fold purification to homogeneity of deoxycytidine kinase from a crude ammonium sulphate fraction of Lactobacillus acidophilus R-26 extract, with 60 % recovery, a striking instance of the power of affinity chromatography. Elution from the column was performed with dCTP, which is a powerful end-product inhibitor for the enzyme and serves to stabilize it. 

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.

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. 

Sulkowski E. 1996. Immobilized metal-ion affinity chromatography imidazole proton pump and chromatographic sequelae I. Proton pump, J. Mol. Recognit., 9, 389-393. 

Activation of cross-linked agaroses with 1,1-carbonyl-di-imidazole yields a matrix for affinity chromatography devoid of additional charged groups/ The activated matrix (Scheme 7) is relatively stable to hydrolysis, but reacts smoothly with A-nucleophiles such as those present in affinity chromatography ligands and leashes. The matrix was treated with 1-aminobutane, 1,6-diamino-hexane, or 6-aminohexanoic acid and then with soybean trypsin inhibitor or 4-aminobenzamidine via a carbodi-imide-mediated reaction, and the products were shown to be useful for the affinity chromatographic purification of trypsin. 

Studies were also carried out using immobilized metal affinity chromatography. A strain of E. coli with histidine on the surface was passed through iminodiacetic acid gel (IDA gel). Cells were captured and could not be eluted with high salt concentrations up to 1.6 M NaCl was tested. However, imidazol or EDTA released the cells with a recovery yield of approximately 80 % [55]. 

Figure 12 Purification of IgG by immobilized metal affinity chromatography (IMAC). The low concentration of imidazole in the loading buffer (buffer A) should enhance the binding specificity and addition of 0.5M NaCI in the buffers may improve the purity of the antibody. Elution can be achieved by reducing pH or by competitive elution with imidazole.

RNA and DNA have been coupled to cellulose with the aid of a water-soluble carbodi-imide and to cellulose phosphate activated by l,r-carbonyldi-imidazole the products were used in the isolation of specific nucleotide sequences by affinity chromatography. ... 

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