PROCEDURE FOR FREEZE-DRYING MOLECULES ADSORBED TO MICA FLAKES
PROCEDURE FOR FREEZE-DRYING MOLECULES ADSORBED TO MICA FLAKES
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DOI：
10.1016/s0022-2836(83)80179-x
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发表时间：
1983-01-01
影响因子：
5.6
通讯作者：
HEUSER, JE
中科院分区：
文献类型：
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作者：
HEUSER, JE
快速冷冻、深蚀刻、旋转复制技术可用于可视化细胞和细胞组分，但不适用于大分子悬浮液。这些不可避免地在深蚀刻过程中结块或塌陷，可能是由于它们从冰转移到真空过程中产生的表面张力。先前的方案试图通过将大分子附着到新鲜切割的云母干燥和复制来克服这种力。本程序的适应，否则实行深蚀刻技术进行了说明。这些分子与云母薄片的水悬浮液混合，然后快速冷冻并冷冻断裂。断裂不可避免地撞击许多云母片的表面，从而足够干净地切割吸附的大分子，以揭示它们内部有趣的亚结构。随后的深蚀刻步骤暴露出大片未断裂的云母，从而揭示出完整的大分子。这些大分子不会被盐沉积物所掩盖，即使它们被冷冻在高渗溶液中，显然是因为压裂步骤几乎去除了所有覆盖的电解质。这些大分子被最低限度地冷冻干燥（因为在-102 ℃下蚀刻仅3分钟后暴露就足够了）。C）因此它们保持其三维拓扑结构。软体动物血蓝蛋白是一种很好的内标。它在稳定的溶液中可商购获得，与所有尺寸的大分子混合良好，并且由显示不同的5-起始表面螺旋的颗粒组成，这些螺旋在过去已经被仔细测量并且具有已知的旋向性，当检查大多数类型的扩展大分子所具有的各种螺旋图案时，可用于确定显微照片的取向。断裂的血蓝蛋白颗粒还显示出特征性的内部结构，这允许确定上述分子裂解的升高。如果这些步骤成为问题，软体动物血蓝蛋白足够脆弱，可以反映出冷冻不良或复制不良。综述了几种大分子的研究进展，包括可溶性酶、抗体、丝状蛋白和核蛋白。这些图像在很大程度上对应于先前通过负染色获得的图像。他们的结构的新的细节被注意到，图像被用来说明新的程序的优点和缺点。
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 during their transfer from ice to vacuum. Previous protocols have attempted to overcome such forces by attaching macromolecules to freshly cleaved mica drying and replication. An adaptation of this procedure to the deep-etch technique as otherwise practiced was described. The molecules were mixed with an aqueous suspension of tiny flakes of mica and then quick-frozen and freeze-fractured. The fracture inevitably strikes the surfaces of many mica flakes and thereby cleaves the adsorbed macromolecules cleanly enough to reveal interesting substructure within them. The subsequent step of deep-etching exposes large expanses of unfractured mica and thus reveals intact macromolecules. These macromolecules are not obscured by salt deposits, even if they were frozen in hypertonic solutions, apparently because the fracturing step removes nearly all of the overlying electrolyte. These macromolecules are minimally freeze-dried (since exposure is sufficient after only 3 min of etching at -102.degree. C) so they retain their 3-dimensional topology. Molluscan hemocyanin is a good internal standard for this new technique. It is available commercially in stable solutions, mixes well with all sizes of macromolecules, and consists of particles that display distinct 5-start surface helices, which have been measured carefully in the past and which possess a known handedness, useful for determining the orientation of micrographs when examining the various helical patterns possessed by most types of extended macromolecules. The fractured hemocyanin particles also display characteristic internal structures, which permit determination of the elevation of the molecular cleavage described above. Molluscan hemocyanin is delicate enough to reflect bad freezing or poor replication, if these steps become a problem. A survey of several macromolecules is presented, including soluble enzymes, antibodies, filamentous proteins and nucleoproteins. These images, for the most part, correspond to those previously obtained by negative staining. New details of their structures are noted, and the images are used to illustrate both the advantages and drawbacks of the new procedure.