Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy
| Autor: | Philipp Selenko, Gerhard Wagner, Zach Serber, Robert Hänsel, Volker Dötsch, Sina Reckel, Frank Löhr, James E. Ferrell |
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| Rok vydání: | 2006 |
| Předmět: |
biology
Macromolecular Substances Xenopus Drug design Transfection Nuclear magnetic resonance spectroscopy Protein engineering biology.organism_classification Oocyte General Biochemistry Genetics and Molecular Biology Xenopus laevis medicine.anatomical_structure Structural biology Biochemistry Isotope Labeling Escherichia coli medicine Biophysics Animals Nuclear Magnetic Resonance Biomolecular Cells Cultured Ovum Macromolecule |
| Zdroj: | Nature Protocols. 1:2701-2709 |
| ISSN: | 1750-2799 1754-2189 |
| Popis: | The noninvasive character of NMR spectroscopy, combined with the sensitivity of the chemical shift, makes it ideally suited to investigate the conformation, binding events and dynamics of macromolecules inside living cells. These 'in-cell NMR' experiments involve labeling the macromolecule of interest with a nonradioactive but NMR-active isotope (15N or 13C). Cellular samples are prepared either by selectively overexpressing the protein in suitable cells (e.g., bacterial cells grown on isotopically labeled media), or by injecting isotopically labeled proteins directly into either cells or cell extracts. Here we provide detailed protocols for in-cell NMR experiments in the prokaryotic organism Escherichia coli, as well as eukaryotic cells and extracts employing Xenopus laevis oocytes or egg extracts. In-cell NMR samples with proteins overexpressed in E. coli can be produced within 13-14 h. Preparing Xenopus oocyte samples for in-cell NMR experiments takes 6-14 h depending on the oocyte preparation scheme and the injection method used. |
| Databáze: | OpenAIRE |
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