Differential extraction of proteins from paraformaldehyde-fixed cells: Lessons from synaptophysin and other membrane proteins
Differential extraction of proteins from paraformaldehyde-fixed cells: Lessons from synaptophysin and other membrane proteins
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DOI：
10.1006/meth.1998.0664
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发表时间：
1998-10-01
期刊：
影响因子：
4.8
通讯作者：
Huttner, WB
中科院分区：
文献类型：
--
作者：
Hannah, MJ;Weiss, U;Huttner, WB
白色使用免疫荧光显微镜研究突触体蛋白在PC 12细胞中的定位，我们注意到其表观亚细胞分布的显著差异取决于是否使用毛地黄皂苷或Triton X-100作为多聚甲醛（PFA）固定的细胞的透化剂。我们发现，这种差异是由于毛地黄皂苷处理的细胞中的表位不可及性结合Triton X-100透化上几乎定量提取的抗原。这两种现象是不同的相对于不同的突触体蛋白含有车厢。从PFA固定的细胞中提取的抗原也与其他膜蛋白一起观察到，但与胞浆蛋白和分泌途径内腔中的蛋白不一起观察到。值得注意的是，从PFA固定的细胞中提取的一些膜蛋白以较高分子量的形式，我们认为这代表了它们在体内的寡聚状态。我们的观察结果的影响进行了讨论相对于免疫荧光显微镜的方法，也可能使用多聚甲醛作为在体内的交联剂的膜蛋白四级结构的研究。(C)北京：科学出版社.
White investigating the localization of synaptophysin in PC12 cells using immunofluorescence microscopy, we noticed a striking difference in its apparent subcellular distribution depending on whether digitonin or Triton X-100 was used as permeabilization agent of paraformaldehyde (PFA)-fixed cells. We found that this difference was due to epitope inaccessibility in the digitonin-treated cells combined with an almost quantitative extraction of the antigen on Triton X-100 permeabilization. Both phenomena were differential with respect to the various synaptophysin-containing compartments. The extraction of antigen from PFA-fixed cells was also seen with other membrane proteins but not with cytosolic proteins and proteins in the lumen of the secretory pathway. Significantly, some of the membrane proteins were extracted from the PFA-fixed cells in higher-molecular-weight forms which we believe represent their in vivo oligomeric states. The implications of our observations are discussed with respect to the method of immunofluorescence microscopy and also to the possible use of paraformaldehyde as an in vivo crosslinker for the study of membrane protein quaternary structure. (C) 1998 Academic Press.