Use of surface affinity enrichment and cryo-embedding to prepare in vitro reconstituted mitotic chromosomes for EM tomography.
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.
König
Peter
P
Department of Biochemistry and Biophysics, The Howard Hughes Medical Institute, University of California, San Francisco, CA 94143-2240, USA.
Braunfeld
Michael
M
Agard
David A
DA
D000818:Animals; D002875:Chromosomes / Q000648:ultrastructure*; D003584:Cytological Techniques / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes; D017110:Freeze Substitution / Q000379:methods; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D008677:Metaphase; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence; D008938:Mitosis; D009865:Oocytes / Q000648:ultrastructure; D016613:Plastic Embedding / Q000379:methods; D014982:Xenopus laevis
D005456:Fluorescent Dyes