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Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures.
Hongxia Fu, and
Benjamin S Freedman, and
Chwee Teck Lim, and
Rebecca Heald, and
Jie Yan
January 2009,
Methods in molecular biology (Clifton, N.J.),
Hongxia Fu, and
Benjamin S Freedman, and
Chwee Teck Lim, and
Rebecca Heald, and
Jie Yan
July 2006,
Journal of microscopy,
Hongxia Fu, and
Benjamin S Freedman, and
Chwee Teck Lim, and
Rebecca Heald, and
Jie Yan
June 2018,
Cold Spring Harbor protocols,
Hongxia Fu, and
Benjamin S Freedman, and
Chwee Teck Lim, and
Rebecca Heald, and
Jie Yan
September 2018,
Cold Spring Harbor protocols,
Hongxia Fu, and
Benjamin S Freedman, and
Chwee Teck Lim, and
Rebecca Heald, and
Jie Yan
June 2008,
Microscopy research and technique,
Hongxia Fu, and
Benjamin S Freedman, and
Chwee Teck Lim, and
Rebecca Heald, and
Jie Yan
October 2013,
Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada,
Hongxia Fu, and
Benjamin S Freedman, and
Chwee Teck Lim, and
Rebecca Heald, and
Jie Yan
February 2019,
Cold Spring Harbor protocols,
Hongxia Fu, and
Benjamin S Freedman, and
Chwee Teck Lim, and
Rebecca Heald, and
Jie Yan
June 2021,
Journal of visualized experiments : JoVE,
Hongxia Fu, and
Benjamin S Freedman, and
Chwee Teck Lim, and
Rebecca Heald, and
Jie Yan
December 2011,
Micron (Oxford, England : 1993),
Hongxia Fu, and
Benjamin S Freedman, and
Chwee Teck Lim, and
Rebecca Heald, and
Jie Yan
October 2006,
Microscopy research and technique,
