A ONE-STEP PROCEDURE FOR BIOTINYLATION AND CHEMICAL CROSS-LINKING OF LYMPHOCYTE SURFACE AND INTRACELLULAR MEMBRANE-ASSOCIATED MOLECULES
A ONE-STEP PROCEDURE FOR BIOTINYLATION AND CHEMICAL CROSS-LINKING OF LYMPHOCYTE SURFACE AND INTRACELLULAR MEMBRANE-ASSOCIATED MOLECULES
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DOI：
10.1006/abio.1995.1054
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发表时间：
1995-01-01
影响因子：
2.9
通讯作者：
PAGLER, EB
中科院分区：
文献类型：
--
作者：
ALTIN, JG;PAGLER, EB
在涉及细胞表面分子标记的研究中，细胞表面生物素化的使用已成为放射性碘化的流行替代方案。通过免疫沉淀、SDS-PAGE 分析和增强化学发光 (ECL) 的蛋白质检测，探索了使用生物素化和化学交联来标记和共价连接 T 淋巴细胞中相关分子的可能性。在同双功能交联剂 DTSSP [3,3'-二硫代-双（磺基琥珀酰亚胺丙酸酯），0.2 mg/ml] 存在下，对来自 1% Triton X-100 小鼠胸腺细胞表面生物素化的裂解物中的 CD45 和 Thy-1 mAb 免疫沉淀物进行还原和非还原 SDS-PAGE 分析，结果表明表面分子 CD45 和 Thy-1 可以生物素化和化学连接，并且与之前使用细胞表面放射性碘化的报告一致。此外，用溶血卵磷脂 (20-25 μ g/ml) 透化细胞后生物素化的鼠 T 细胞克隆 D10 和人白血病 T 细胞系 Jurkat 的 1% 毛地黄皂苷裂解物中的 CD45 和 CD3 mAb 进行免疫沉淀，揭示了几种额外的 16、32、34、56、60 和 80 kDa 的共沉淀分子不 在表面生物素化细胞裂解物的免疫沉淀物中检测到。在表面生物素化细胞的裂解物中未能检测到这些分子表明这些分子通过细胞表面标记的生物素化效果较差，或者位于细胞内。其中一些分子的细胞内定位得到以下事实的支持：在溶血卵磷脂存在下生物素化的 Jurkat 细胞裂解物的 CD45 mAb 免疫沉淀物中看到的 56-kDa 分子可以通过 p56(lck) Ab 的免疫印迹鉴定为 src 样酪氨酸激酶 p56(lck)。因此，结果表明，某些相关的细胞表面和细胞内蛋白质可以通过一步程序进行生物素化和化学连接。我们预计该技术将进一步扩大生物素化和非放射性蛋白质检测的用途，特别是在表征细胞表面受体与细胞表面或细胞内部其他分子的分子关联的研究中。 (C) 1995 学术出版社
The use of cell surface biotinylation has become a popular alternative to radioiodination in studies involving labeling of cell surface molecules. The possibility of using biotinylation and chemical cross-linking to label and covalently link associated molecules in T lymphocytes was explored using immunoprecipitation, SDS-PAGE analysis, and protein detection by enhanced chemiluminescence (ECL). Reduced and nonreduced SDS-PAGE analysis of CD45 and Thy-1 mAb immunoprecipitates from 1% Triton X-100 lysates of murine thymocytes surface biotinylated in the presence of the homobifunctional cross-linker DTSSP [3,3'-dithio-bis (sulfosuccinimidylpropionate), 0.2 mg/ml] revealed that the surface molecules CD45 and Thy-1 can be biotinylated and chemically linked, and is consistent with a previous report using cell surface radioiodination. Also, CD45 and CD3 mAb immunoprecipitates from 1% digitonin lysates of the murine T cell clone D10 and of the human leukemic T cell line Jurkat, biotinylated after permeabilization of the cells with lysolecithin (20-25 mu g/ml), revealed several additional coprecipitating molecules of 16, 32, 34, 56, 60 and 80 kDa not detected in immunoprecipitates from lysates of surface-biotinylated cells. The failure to detect these molecules in lysates of surface-biotinylated cells suggests that these molecules biotinylate poorly by cell surface labeling, or are localized intracellularly. The intracellular localization of some of these molecules is supported by the fact that the 56-kDa molecule seen in CD45 mAb immunoprecipitates from lysates of Jurkat cells biotinylated in the presence of lysolecithin could be identified as the src-like tyrosine kinase p56(lck) by immunoblotting with p56(lck) Abs. The results show, therefore, that certain associated cell surface and intracellular proteins can be biotinylation and chemically linked using a single-step procedure. We expect that the technique will further broaden the use of biotinylation and nonradioactive protein detection, particularly in studies characterizing the molecular associations of cell surface receptors with other molecules, either on the cell surface or inside the cell. (C) 1995 Academic Press, Inc.