ABSTRACT
Use of Immobilized-Metal Affinity...................................................... 259 10.2 Experimental Considerations .............................................................................. 260
10.2.1 Support Selection.................................................................................. 260 10.2.2 Choice of Chelating Agents and Adsorption Center ........................... 261 10.2.3 Selection of Metal Ions ........................................................................ 262 10.2.4 Mobile-Phase Selection ........................................................................ 265 10.2.5 Packing and Preparing IMAC Columns............................................... 266 10.2.6 Elution Strategies.................................................................................. 266
10.2.6.1 pH Elution Schemes............................................................ 266 10.2.6.2 Addition of Competing Agents ........................................... 267
10.3 Applications......................................................................................................... 267 10.3.1 Protein Purification by IMAC .............................................................. 268
10.3.1.1 Proteins with Surface Histidines......................................... 268 10.3.1.2 His-Tagged Proteins ............................................................ 269 10.3.1.3 Plant Proteins....................................................................... 269
10.3.2 Studies of Protein Surface Topography and Posttranslational Modifications ..................................................... 270
10.3.3 Microanalytical Methods ...................................................................... 271 10.3.3.1 Immobilized Metal-Ion Affinity Gel Electrophoresis......... 272 10.3.3.2 Immobilized Metal-Ion Affinity
Capillary Electrophoresis .................................................... 273 10.3.3.3 Immobilized Metal-Ion Affinity Partitioning...................... 275
10.3.4 Applications of IMAC in Genomics and Postgenomic Studies .......... 275 10.4 Summary and Conclusions.................................................................................. 277 Acknowledgments........................................................................................................... 277 Symbols and Abbreviations ............................................................................................ 277 References ....................................................................................................................... 278
10.1 INTRODUCTION
The idea of using immobilized metal-chelate complexes in ligand-exchange chromatography for the separation of amino acids, nucleic acids, peptides, and proteins was developed in the early 1960s [1-3]. In 1975, Porath and coworkers [4] introduced immobilized metal-ion affinity chromatography (IMAC) as a purification technique for biomolecules. IMAC is based on the solution-phase interactions of chelated metal ions with proteins and other biological compounds. Other terms used to describe this method are metal-chelate chromatography and metal-ion interaction chromatography.
