Abstract
Immobilized metal affinity chromatography (IMAC) is widely used for purification of proteins, especially "hexahistidine-tagged" recombinant proteins. We previously demonstrated the application of IMAC to selective capture of nucleic acids, including RNA, selectively-denatured genomic DNA, and PCR primers through interactions with purine bases exposed in single-stranded regions. We also found that the binding affinity of nucleic acids for IMAC adsorbents can be increased several-fold by addition of 20 volume% of neutral additives such as ethanol or DMSO. In the present work, it is demonstrated that bound nucleic acids can be effectively eluted with water instead of the usual imidazole-containing competitive eluants, when the surface density of negative charges is enhanced by operation at alkaline pH, or by deliberate metal-underloading of the anionic chelating ligands. With enhanced negative surface charge density, nucleic acid adsorption can be made strongly dependent on the presence of adsorption-promoting additives and/or repulsion-shielding salts, and removal of these induces elution. Complete water-elutability is demonstrated for baker's yeast RNA bound to 10% Cu(II)- underloaded IDA Chelating Sepharose in a binding buffer of 20 mM HEPES, 240 mM NaCl, pH7. Water elutability will significantly enhance the utility of IMAC in nucleic acid separations.
Original language | English (US) |
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Pages (from-to) | 348-353 |
Number of pages | 6 |
Journal | Journal of Molecular Recognition |
Volume | 19 |
Issue number | 4 |
DOIs | |
State | Published - Jul 2006 |
Keywords
- Adsorption isotherm
- Cu(II)-IDA
- IMAC
- Metal chelate affinity
- RNA purification
- Water elution
ASJC Scopus subject areas
- Biochemistry
- Genetics
- Computer Vision and Pattern Recognition
- Immunology
- Molecular Biology