Effects of detergents on the redistribution of gangliosides and GPI-anchored proteins in brain tissue sections
Effects of detergents on the redistribution of gangliosides and GPI-anchored proteins in brain tissue sections
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DOI：
10.1369/jhc.7a7195.2007
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发表时间：
2007-08-01
影响因子：
3.2
通讯作者：
Lauc, Gordan
中科院分区：
文献类型：
--
作者：
Heffer-Lauc, Marija;Viljetic, Barbara;Lauc, Gordan
神经节苷和糖基磷脂酰肌醇（GPI）锚定的蛋白质含有脂质尾巴，将它们拴在细胞膜的外侧。这种与细胞膜的结合模式使它们能够参与脂筏的组织，但它也允许神经节苷脂和gpi锚定蛋白从一个细胞主动释放并插入到另一个细胞的膜中。最近，我们报道了在脂筏分离条件下，Triton X-100引起神经节苷脂和gpi锚定蛋白的显著重新分布。为了寻找一种破坏性较小的清洁剂，我们评估了CHAPS、皂苷、脱氧胆酸、Trappsol、Tween 20、Triton X-100、Brij 96V、Brij 98和SIDS对脑组织切片的影响。在室温下，所有洗涤剂（浓度为1%）都能提取大量的神经节苷脂和Thy-1。在4C时，提取较弱，但Triton X-100、CHAPS和脱氧胆酸导致GD1a和Thy-1从灰质向白质重新分布。当切片在一抗存在下用清洁剂孵育时，再分配和提取都显着增加。在接受测试的九种洗涤剂中，没有一种是理想的选择。然而，Brij 96V似乎能够充分显示髓磷脂表位，同时引起最少的伪影。该手稿包含在线补充材料http://www.jhc.org。请在线访问本文以查看这些材料。
Gangliosides and glycosylphosphatidylinositol (GPI)-anchored proteins contain lipid tails that tether them to the outer side of the cell membrane. This mode of association with the cell membrane enables them to take part in the organization of lipid rafts, but it also permits gangliosides and GPI-anchored proteins to be actively released from one cell and inserted into the membrane of another cell. Recently, we reported that under conditions of lipid raft isolation, Triton X-100 causes significant redistribution of both gangliosides and GPI-anchored proteins. Aiming to find a less disruptive detergent, we evaluated the effects of CHAPS, Saponin, deoxycholic acid, Trappsol, Tween 20, Triton X-100, Brij 96V, Brij 98, and SIDS on brain tissue sections. At room temperature, all detergents (1% concentration) extracted significant amounts of both gangliosides and Thy-1. At 4C, the extraction was weaker, but Triton X-100, CHAPS, and deoxycholic acid caused significant redistribution of GD1a and Thy-1 from gray matter into the white matter. Both redistribution and extraction were significantly augmented when sections were incubated with detergents in the presence of primary antibodies. Of the nine tested detergents, none is the ideal choice. However, Brij 96V appears to be able to sufficiently reveal myelin epitopes while causing the least amount of artifacts. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.