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.
CITATION STYLE
Clubbs Coldron, A. K. M., Byrne, D. P., & Eyers, P. A. (2020). Analysis of 1- and 3-Phosphohistidine (pHis) Protein Modification Using Model Enzymes Expressed in Bacteria. In Methods in Molecular Biology (Vol. 2077, pp. 63–81). Humana Press Inc. https://doi.org/10.1007/978-1-4939-9884-5_5
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