Analysis of 1- and 3-Phosphohistidine (pHis) Protein Modification Using Model Enzymes Expressed in Bacteria

  • Alice K. M. Clubbs Coldron
  • Dominic P. Byrne
  • Patrick A. EyersEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2077)


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.

Key words

NME1 NME2 NDPK-A NDPK-B PGAM1 Histidine phosphorylation Western blotting Immunoblotting Differential scanning fluorimetry N1-phosphohistidine N3-phosphohistidine 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Alice K. M. Clubbs Coldron
    • 1
  • Dominic P. Byrne
    • 1
  • Patrick A. Eyers
    • 1
    Email author
  1. 1.Department of Biochemistry, Institute of Integrative BiologyUniversity of LiverpoolLiverpoolUK

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