Detection of membrane-anchoring lipid modifications of proteins in cells by radioactive metabolic labeling.
Detection of membrane-anchoring lipid modifications of proteins in cells by radioactive metabolic labeling.
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DOI：
10.1016/j.xpro.2023.102416
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发表时间：
2023-09-15
期刊：
影响因子：
--
通讯作者：
Kuchay, Shafi
中科院分区：
文献类型：
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作者：
Ahmed, Sameer;Ferguson, Rachel;Adhikari, Ritika;Kuchay, Shafi
异戊二烯化和棕榈酰化是细胞蛋白质的两种主要脂质修饰，将蛋白质锚定在细胞膜上。在这里，我们提出了一种通过放射性代谢标记检测细胞蛋白质中这些修饰的方案。我们描述了细胞代谢标记、收获细胞进行免疫沉淀、对免疫复合物进行 SDS-PAGE 以及将其转移到聚偏氟乙烯 (PVDF) 膜的步骤。然后，我们通过将 PVDF 膜暴露于荧光屏并使用荧光成像仪来详细检测标记的目标蛋白。有关该协议的完整详细信息，请参阅Liang 等人。使用氚代谢物标记进行细胞蛋白异戊二烯化和棕榈酰化检测高度灵敏且可重复的实验方案适用于内源性和外源性表达蛋白质的实验方案出版商注：进行任何实验方案都需要遵守当地实验室安全和道德机构指南。异戊二烯化和棕榈酰化是细胞蛋白质的两种主要脂质修饰，将蛋白质锚定在细胞膜上。在这里，我们提出了一种通过放射性代谢标记检测细胞蛋白质中这些修饰的方案。我们描述了细胞代谢标记、收获细胞进行免疫沉淀、对免疫复合物进行 SDS-PAGE 以及将其转移到聚偏氟乙烯 (PVDF) 膜的步骤。然后，我们通过将 PVDF 膜暴露于荧光屏并使用荧光成像仪来详细检测标记的目标蛋白。
Prenylation and palmitoylation are two major lipid modifications of cellular proteins that anchor proteins to cell membranes. Here, we present a protocol for detecting these modifications in cellular proteins by radioactive metabolic labeling. We describe steps for metabolic labeling of cells, cell harvesting for carrying out immunoprecipitations, subjecting immunocomplexes to SDS-PAGE, and transferring them to polyvinylidine flouride (PVDF) membranes. We then detail detection of labeled target proteins by exposing PVDF membranes to phosphor screens and using a phosphor imager machine. For complete details of this protocol, please refer to Liang et al. Cellular protein prenylation and palmitoylation detection with tritiated-metabolite labeling Highly sensitive and reproducible protocols Protocols adaptable for endogenous and exogenously expressed proteins Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Prenylation and palmitoylation are two major lipid modifications of cellular proteins that anchor proteins to cell membranes. Here, we present a protocol for detecting these modifications in cellular proteins by radioactive metabolic labeling. We describe steps for metabolic labeling of cells, cell harvesting for carrying out immunoprecipitations, subjecting immunocomplexes to SDS-PAGE, and transferring them to polyvinylidine flouride (PVDF) membranes. We then detail detection of labeled target proteins by exposing PVDF membranes to phosphor screens and using a phosphor imager machine.
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影响因子：
8.8
作者：
通讯作者：
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影响因子：
16.8
作者：
Kuchay, Shafi;Wang, Hui;Pagano, Michele
通讯作者：
Pagano, Michele
影响因子：
16
作者：
Wang, CF;Gale, M;Ye, J
通讯作者：
Ye, J
用氚化棕榈酸酯对 Ras 进行代谢标记，以监测棕榈酰化和去棕榈酰化。
DOI：
10.1007/978-1-62703-791-4_3
发表时间：
2014
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Tsai, Frederick D;Wynne, Joseph P;Ahearn, Ian M;Philips, Mark R
通讯作者：
Philips, Mark R
影响因子：
14.8
作者：
通讯作者：
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