| Water-elutability of nucleic acids from metal-chelate affinity adsorbents: enhancement by control of surface charge density. | |
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MedLine Citation:
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PMID: 16865664 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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, pH 7. Water elutability will significantly enhance the utility of IMAC in nucleic acid separations. |
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Authors:
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Joseph Y Fu; Ajish S R Potty; George E Fox; Richard C Willson |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S. |
Journal Detail:
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Title: Journal of molecular recognition : JMR Volume: 19 ISSN: 0952-3499 ISO Abbreviation: J. Mol. Recognit. Publication Date: 2006 Jul-Aug |
Date Detail:
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Created Date: 2006-08-28 Completed Date: 2006-09-29 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 9004580 Medline TA: J Mol Recognit Country: England |
Other Details:
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Languages: eng Pagination: 348-53 Citation Subset: IM |
Copyright Information:
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Copyright 2006 John Wiley & Sons, Ltd. |
Affiliation:
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Department of Chemical Engineering, University of Houston, Houston, TX 77204-4004, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Adsorption Chelating Agents / chemistry* Chromatography, Affinity Copper / chemistry* Hydrogen-Ion Concentration Imidazoles / chemistry Imino Acids / chemistry Ligands Oligonucleotides / chemistry*, isolation & purification Purines / chemistry RNA, Fungal / chemistry* Sepharose / chemistry Surface Properties Water / chemistry* |
| Chemical | |
Reg. No./Substance:
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0/Chelating Agents; 0/Imidazoles; 0/Imino Acids; 0/Ligands; 0/Oligonucleotides; 0/Purines; 0/RNA, Fungal; 142-73-4/iminodiacetic acid; 288-32-4/imidazole; 7440-50-8/Copper; 7732-18-5/Water; 9012-36-6/Sepharose |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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