Chakrabandhu Krittalak, Huault Sébastien, Hueber Anne-Odile
Université Côte d'Azur, Institut de Biologie Valrose, CNRS UMR 7277, Inserm UMR 1091, Parc Valrose, Bâtiment des Sciences Naturelles, 06108, Nice, France.
Institut de Biologie Valrose, CNRS UMR 7277, INSERM UMR,Université de Nice, Nice, France.
Methods Mol Biol. 2017;1557:173-188. doi: 10.1007/978-1-4939-6780-3_16.
Phosphorylation of two tyrosines in the death domain of CD95 is a critical mechanism in determining the receptor's choices between cell death and survival signals. Recently, site-specific monoclonal antibodies against phosphorylated tyrosines of CD95 have been generated and used to successfully detect each phosphorylated death domain tyrosine of CD95 directly and separately by immunoblotting. Here we provide detailed protocols and useful tips for a successful site-specific detection of phosphorylated death domain tyrosine of CD95 following a protein separation by sizes (conventional SDS-PAGE) and by degrees of phosphorylation (phospho-protein affinity, mobility shift SDS-PAGE).
CD95死亡结构域中两个酪氨酸的磷酸化是决定该受体在细胞死亡和生存信号之间做出选择的关键机制。最近，已制备出针对CD95磷酸化酪氨酸的位点特异性单克隆抗体，并用于通过免疫印迹直接且分别成功检测CD95每个磷酸化死亡结构域酪氨酸。本文我们提供了详细方案和实用技巧，以便在按大小（传统SDS-PAGE）和磷酸化程度（磷酸化蛋白亲和、迁移率变动SDS-PAGE）进行蛋白质分离后，成功对CD95磷酸化死亡结构域酪氨酸进行位点特异性检测。
