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SDS-PAGE and Dot Blot Autoradiography: Tools for Quantifying Histidine Kinase Autophosphorylation

  • Jonathan T. Fischer
  • Ilana Heckler
  • Elizabeth M. BoonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2077)

Abstract

Histidine kinases play a vital role in bacterial signal transduction. However, methods for studying the activity of histidine kinases in vitro are limited in comparison to those for investigating serine, threonine, and tyrosine kinases, largely due to the lability of the phosphoramidate (P-N) bond. Here, we describe two useful methods for quantifying histidine kinase autophosphorylation: SDS-PAGE autoradiography and dot blot autoradiography/scintillation counting.

Key words

Quantification Histidine kinase Autophosphorylation Autoradiography SDS-PAGE Dot blot Two-component signaling 

Notes

Acknowledgments

This work was supported by NIH GM118894 and NSF CHE-1607532.

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Copyright information

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

Authors and Affiliations

  • Jonathan T. Fischer
    • 1
  • Ilana Heckler
    • 1
  • Elizabeth M. Boon
    • 1
    • 2
    Email author
  1. 1.Department of ChemistryStony Brook UniversityStony BrookUSA
  2. 2.Institute of Chemical Biology and Drug DiscoveryStony BrookUSA

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