Iancu, Cristina V. and Tivol, William F. and Schooler, Jordan B. and Dias, D. Prabha and Henderson, Gregory P. and Murphy, Gavin E. and Wright, Elizabeth R. and Li, Zhuo and Yu, Zhiheng and Briegel, Ariane and Gan, Lu and He, Yongning and Jensen, Grant J. (2006) Electron cryotomography sample preparation using the Vitrobot. Nature Protocols, 1 (6). pp. 2813-2819. ISSN 1754-2189 http://resolver.caltech.edu/CaltechAUTHORS:20110803-133119203
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Electron cryotomography is the highest-resolution structural technique currently available that can be applied to unique objects such as flexible large protein complexes, irregular viruses, organelles and small cells. Specimens are preserved in a near-native, 'frozen-hydrated' state by vitrification. The thickness of the vitreous ice must be optimized for each specimen, and gold fiducials are typically added to facilitate image alignment. Here, we describe in detail our protocols for electron cryotomography sample preparation including (i) introduction of fiducial markers into the sample and (ii) sample vitrification. Because we almost exclusively use an automated, climate-controlled plunge-freezing device (the FEI Vitrobot) to vitrify our samples, we discuss its operation and parameters in detail. A session in which eight grids are prepared takes 1.5–2 h.
|Additional Information:||© 2006 Nature Publishing Group. Published online: 18 January 2007. Competing interests statement: The authors declare no competing financial interests.|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||George Porter|
|Deposited On:||03 Aug 2011 20:56|
|Last Modified:||03 Aug 2011 20:56|
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