Procedure for freeze-drying molecules adsorbed to mica flakes

摘要: The quick-freeze, deep-etch, rotary-replication technique is useful for visualizing cells and cell fractions but does not work with suspensions of macromolecules. These inevitably clump or collapse during deep-etching, presumably due to surface tension forces that develop their transfer from ice vacuum. Previous protocols have attempted overcome such by attaching macromolecules freshly cleaved mica before drying replication. I describe here an adaptation this procedure the deep-etch as otherwise practiced. My innovation mix molecules aqueous suspension tiny flakes then quick-freeze freeze-fracture exactly if one were dealing cells. fracture strikes surfaces many thereby cleaves adsorbed cleanly enough reveal interesting substructure within them. subsequent step deep-etching exposes large expanses unfractured thus reveals intact are obscured salt deposits, even they frozen in hypertonic solutions, apparently because fracturing removes nearly all overlying electrolyte. Moreover, these minimally freeze-dried (since exposure sufficient after only 3 min etching at -102 degrees C) so retain three-dimensional topology. show molluscan hemocyanin a good internal standard new technique. It available commercially stable mixes well sizes macromolecules, consists particles display distinct five-start helices, which been measured carefully past possess known handedness, determining orientation micrographs when examining various helical patterns possessed most types extended fractured also characteristic structures, permit determination elevation "molecular cleavage" described above. Finally, delicate reflect bad freezing poor replication, steps become problem. A survey several presented, including soluble enzymes, antibodies, filamentous proteins nucleoproteins. images, part, correspond those previously obtained negative staining. New details structures noted, images used illustrate both advantages drawbacks procedure.