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

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


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 



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


  1. 1.
    Parkinson JS, Kofoid EC (1992) Communication modules in bacterial signaling proteins. Annu Rev Genet 26:71–112CrossRefGoogle Scholar
  2. 2.
    Stock AM, Robinson VL, Goudreau PN (2000) Two-component signal transduction. Annu Rev Biochem 69:183–215CrossRefGoogle Scholar
  3. 3.
    Jung K, Fried L, Behr S, Heermann R (2012) Histidine kinases and response regulators in networks. Curr Opin Microbiol 15:118–124CrossRefGoogle Scholar
  4. 4.
    Szurmant H, Bu L, Brooks CL, Hoch JA (2008) An essential sensor histidine kinase controlled by transmembrane helix interactions with its auxiliary proteins. Proc Natl Acad Sci U S A 105:5891–5896CrossRefGoogle Scholar
  5. 5.
    Hossain S, Boon EM (2017) Discovery of a novel nitric oxide binding protein and nitric oxide-responsive signaling pathway in Pseudomonas aeruginosa. ACS Infect Dis 3:454–461CrossRefGoogle Scholar
  6. 6.
    Arora DP, Boon EM (2012) Nitric oxide regulated two-component signaling in Pseudoalteromonas atlantica. Biochem Biophys Res Commun 421:521–526CrossRefGoogle Scholar
  7. 7.
    Price MS, Chao LY, Marletta MA (2007) Shewanella oneidensis MR-1 H-NOX regulation of a histidine kinase by nitric oxide. Biochemistry 46:13677–13683CrossRefGoogle Scholar
  8. 8.
    Nixon BT, Ronson CW, Ausubel FM (1986) Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc Natl Acad Sci U S A 83:7850–7854CrossRefGoogle Scholar
  9. 9.
    Ronson CW, Nixon BT, Ausubel FM (1987) Conserved domains in bacterial regulatory proteins that respond to environmental stimuli. Cell 49:579–581CrossRefGoogle Scholar
  10. 10.
    Uhl MA, Miller JF (1996) Integration of multiple domains in a two-component sensor protein: the Bordetella pertussis BvgAS phosphorelay. EMBO J 15:1028–1036CrossRefGoogle Scholar
  11. 11.
    Gallo R, Provenzano C, Carbone R, Di Fiore PP, Castellani L, Falcone G, Alemà S (1997) Regulation of the tyrosine kinase substrate Eps8 expression by growth factors, v-Src and terminal differentiation. Oncogene 15:1929–1936CrossRefGoogle Scholar
  12. 12.
    Wei Y, Yu L, Bowen J, Gorovsky MA, Allis CD (1999) Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell 97:99–109CrossRefGoogle Scholar
  13. 13.
    Sarg B, Helliger W, Talasz H, Förg B, Lindner HH (2006) Histone H1 phosphorylation occurs site-specifically during interphase and mitosis: identification of a novel phosphorylation site on histone H1. J Biol Chem 281:6573–6580CrossRefGoogle Scholar
  14. 14.
    Stock JB, Stock AM, Mottonen JM (1990) Signal transduction in bacteria. Nature 344:395–400CrossRefGoogle Scholar
  15. 15.
    Kee JM, Muir TW (2012) Chasing phosphohistidine, an elusive sibling in the phosphoamino acid family. ACS Chem Biol 7:44–51CrossRefGoogle Scholar
  16. 16.
    Ueno TB, Johnson RA, Boon EM (2015) Optimized assay for the quantification of histidine kinase autophosphorylation. Biochem Biophys Res Commun 465:331–337CrossRefGoogle Scholar

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

Personalised recommendations