Empirical Evidence of Cellular Histidine Phosphorylation by Immunoblotting Using pHis mAbs
Empirical Evidence of Cellular Histidine Phosphorylation by Immunoblotting Using pHis mAbs
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DOI：
10.1007/978-1-4939-9884-5_12
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发表时间：
2020-01-01
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影响因子：
--
通讯作者：
Hunter, Tony
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文献类型：
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作者：
Kalagiri, Rajasree;Adam, Kevin;Hunter, Tony
免疫印迹是一种普遍存在的免疫学技术，有助于检测和定量蛋白质（包括丰度较低的蛋白质）及其翻译后修饰，如磷酸化、乙酰化、泛素化和类小泛素化。该技术包括在SDS-PAGE凝胶上分离蛋白质，将它们转移到PVDF（或硝酸纤维素）膜上，并用特异性抗体进行探测。在这里，我们描述了一种免疫印迹技术检测细胞磷酸组氨酸，不稳定的翻译后修饰，通过优化实验条件，使不稳定的磷酸组氨酸信号是保守的整个实验。
Immunoblotting is a ubiquitous immunological technique that aids in detecting and quantifying proteins (including those of lower abundance) and their posttranslational modifications such as phosphorylation, acetylation, ubiquitylation, and sumoylation. The technique involves electrophoretically separating proteins on an SDS-PAGE gel, transferring them onto a PVDF (or nitrocellulose) membrane and probing with specific antibodies. Here we describe an immunoblotting technique for detecting cellular phosphohistidine, a labile posttranslational modification, by optimizing experimental conditions such that the labile phosphohistidine signal is conserved throughout the experiment.