Clubbs Coldron Alice K M, Byrne Dominic P, Eyers Patrick A
Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, UK.
Methods Mol Biol. 2020;2077:63-81. doi: 10.1007/978-1-4939-9884-5_5.
Despite the discovery of protein histidine (His) phosphorylation nearly six decades ago, difficulties in measuring and quantifying this unstable post-translational modification (PTM) have limited its mechanistic analysis in prokaryotic and eukaryotic signaling. Here, we describe reliable procedures for affinity purification, cofactor-binding analysis and antibody-based detection of phosphohistidine (pHis), on the putative human His kinases NME1 (NDPK-A) and NME2 (NDPK-B) and the glycolytic phosphoglycerate mutase PGAM1. By exploiting isomer-specific monoclonal N1-pHis and N3-pHis antibodies, we describe robust protocols for immunological detection and isomer discrimination of site-specific pHis, including N3-pHis on His 11 of PGAM1.
尽管近六十年前就发现了蛋白质组氨酸（His）磷酸化，但测量和量化这种不稳定的翻译后修饰（PTM）的困难限制了其在原核和真核信号传导中的机制分析。在这里，我们描述了用于亲和纯化、辅因子结合分析以及基于抗体检测磷酸组氨酸（pHis）的可靠程序，该程序针对推定的人类组氨酸激酶NME1（NDPK-A）和NME2（NDPK-B）以及糖酵解磷酸甘油酸变位酶PGAM1。通过利用异构体特异性单克隆N1-pHis和N3-pHis抗体，我们描述了用于位点特异性pHis免疫检测和异构体区分的强大方案，包括PGAM1第11位组氨酸上的N3-pHis。
