Journal
NATURE PROTOCOLS
Volume 3, Issue 2, Pages 288-302Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2007.533
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Funding
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [P01GM066275] Funding Source: NIH RePORTER
- NIGMS NIH HHS [P01-GM066275] Funding Source: Medline
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Hydroxyl radical (center dot OH) footprinting provides comprehensive site-specific quantitative information about the structural changes associated with macromolecular folding, interactions and ligand binding. 'Fast Fenton' footprinting is a laboratory-based method for time-resolved. OH footprinting capable of millisecond time resolution readily applicable to DNA and RNA. This protocol utilizes inexpensive chemical reagents (H2O2, Fe(NH4)(2)(SO4)(2), EDTA, thiourea or ethanol) and widely available quench-flow mixers to reveal transient, often short-lived, intermediate states of complex biochemical processes. We describe a protocol developed to study RNA folding that can be readily tailored to particular applications. Once familiar with quench-flow mixer operation and its calibration, nucleic acid labeling and the conduct of a dose-response experiment, a single kinetic experiment of 30 time points takes about 1 h to perform. Sample processing and separation of the. OH reaction products takes several hours. Data analysis can take 45 min to several weeks depending on the depth of analysis conducted.
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