Schlaepfer W W, Freeman L A
J Cell Biol. 1978 Sep;78(3):653-62. doi: 10.1083/jcb.78.3.653.
Intact neurofilaments were isolated in parallel from rat peripheral nerve and spinal cord by osmotic shock into hypotonic media containing divalent cation chelators. Isolated neurofilaments were washed and separated by multiple centrifugations in 0.1 M NaCl. Abundant intact neurofilaments were identified in the washed pellets by negative staining techniques. Their origin from neurofilaments was confirmed by immune electron microscopy. Washed neurofilaments were extracted from lipid and membranous components with 8 M urea. Analyses of neurofilament isolates on sodium dodecyl sulfate gels showed that proteins of 200,000, 150,000, and 69,000 mol wt were the major components of intact neurofilaments derived from rat peripheral and central nervous systems. These same proteins were identified in whole tissue homogenates of both sources and became enriched during the isolation of intact neurofilaments. A minor component of 64,000 mol wt arose during isolation. Other proteins were identified as contaminants. Small amounts of proteins with electrophoretic migration of tubulin and actin remain in neurofilament isolates.
通过渗透休克法，将大鼠外周神经和脊髓中的完整神经丝同时分离到含有二价阳离子螯合剂的低渗介质中。分离出的神经丝经洗涤后，在0.1M NaCl中多次离心分离。通过负染色技术在洗涤后的沉淀中鉴定出大量完整的神经丝。免疫电子显微镜证实了它们来源于神经丝。用8M尿素从脂质和膜成分中提取洗涤后的神经丝。对十二烷基硫酸钠凝胶上的神经丝分离物进行分析表明，分子量为200,000、150,000和69,000的蛋白质是源自大鼠外周和中枢神经系统的完整神经丝的主要成分。在这两种来源的全组织匀浆中都鉴定出了相同的蛋白质，并且在完整神经丝的分离过程中这些蛋白质得到了富集。在分离过程中出现了一种分子量为64,000的次要成分。其他蛋白质被鉴定为污染物。神经丝分离物中仍保留有少量具有微管蛋白和肌动蛋白电泳迁移特征的蛋白质。
