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Ultramicroscopy 103 (2005) 261274 Use of surface affinity enrichment and cryo-embedding
 

Summary: Ultramicroscopy 103 (2005) 261­274
Use of surface affinity enrichment and cryo-embedding
to prepare in vitro reconstituted mitotic chromosomes
for EM tomography
Peter Ko¨ nig, Michael Braunfeld, David A. AgardÃ
Department of Biochemistry and Biophysics, The Howard Hughes Medical Institute, University of California, San Francisco,
CA 94143-2240, USA
Received 3 September 2004; received in revised form 22 November 2004; accepted 9 December 2004
Abstract
We present a novel preparation method for studies of in vitro reconstituted mitotic chromosomes from Xenopus laevis
egg extracts. This method involves a gentle adsorption of chromosomes from the extracts using surface affinity
enrichment, followed by plunge freezing, freeze-substitution and cryo-embedding before examination by EM
tomography. For comparison, chromosomes were also prepared by a conventional method, which included
immobilization of chromosomes in agarose and a room-temperature dehydration/embedding protocol. Three-
dimensional reconstructions showed that samples prepared with the new method have a greater interconnectivity of
sub-structural features and a higher compaction ratio together with an apparently less perturbed chromatin structure
than those prepared using the conventional approach. The implications of the new method for the preparation of other
difficult samples and additional application possibilities are discussed.
r 2005 Elsevier B.V. All rights reserved.
PACS: 87.64.D; 06.60.E

  

Source: Agard, David - Department of Biochemistry and Biophysics, University of California at San Francisco

 

Collections: Biotechnology; Biology and Medicine