Nickel-chelate affinity chromatography

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 

Elution of the bound antibody-enzyme conjugate occurs by only a slight shift in pH to acidic conditions or through the inclusion of a metal-chelating agent like EDTA or imidazole in the binding buffer. Either method of elution is mild compared to most immunoaffinity separation techniques (discussed in the previous section). Thus, purification of the antibody-enzyme complex can be done without damage to the activity of either component. 

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. 

Dissolve 50 mg of nickel ammonium sulfate per ml of deionized water. Apply 1ml of nickel solution per ml of gel to the column. Note The metal salt and all solutions containing it should be considered hazardous waste and disposed of according to relevant environmental regulations. 

Wash the column with 10 volumes of water, then equilibrate the support with 2 volumes of 10 mM sodium phosphate, 0.15 M NaCl, pH 7.0 (binding buffer). 

Pack a column containing an immobilized iminodiacetic acid support (Pierce). The column size should be no less than 1.5 times that required to bind the anticipated amount of conjugate to be applied. The maximal capacity of such a column for binding antibody can be up to 50 mg/ml gel however, best results are obtained if no more than 10—20 mg/ml of conjugate is applied. 

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Figure 20.15 An affinity chromatography support containing iminodiacetic acid groups chelated with nickel may be used to remove excess enzyme after reactions to produce antibody-enzyme conjugates. The nickel chelate binds to the antibody in the Fc region, retaining the conjugate while allowing free enzyme to pass through the gel unretarded.

Finally, it should be pointed out that until now the application of ion chromatography to the analysis of heavy and transition metals such as iron, copper, nickel, and manganese in the ppb range in 50% sodium hydroxide solution [96, 97] was not possible. A concentration procedure developed by Kingston et al. [98] is used for the AAS analysis of heavy metals in sea water. It is based on the selective concentration of heavy metals on a macroporous iminodiacetic acid resin, where divalent cations are retained by chelation. Their affinity toward the stationary phase decreases in the order... 

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