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

Jonathan T Fischer; Ilana Heckler; Elizabeth M Boon

doi:10.1007/978-1-4939-9884-5_3

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

Methods Mol Biol. 2020:2077:37-49. doi: 10.1007/978-1-4939-9884-5_3.

Authors

Jonathan T Fischer¹, Ilana Heckler¹, Elizabeth M Boon^{2

3}

Affiliations

¹ Department of Chemistry, Stony Brook University, Stony Brook, NY, USA.
² Department of Chemistry, Stony Brook University, Stony Brook, NY, USA. [email protected].
³ Institute of Chemical Biology and Drug Discovery, Stony Brook, NY, USA. [email protected].

PMID: 31707650
DOI: 10.1007/978-1-4939-9884-5_3

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.

Keywords: Autophosphorylation; Autoradiography; Dot blot; Histidine kinase; Quantification; SDS-PAGE; Two-component signaling.

Publication types

Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Autoradiography* / methods
Bacterial Proteins / metabolism
Electrophoresis, Polyacrylamide Gel* / methods
Histidine Kinase / chemistry
Histidine Kinase / metabolism*
Immunoblotting* / methods
Phosphorylation
Signal Transduction

Substances

Bacterial Proteins
Histidine Kinase

Grants and funding

R01 GM118894/GM/NIGMS NIH HHS/United States