SDS-PAGE and Dot Blot Autoradiography: Tools for Quantifying Histidine Kinase Autophosphorylation
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 wordsQuantification Histidine kinase Autophosphorylation Autoradiography SDS-PAGE Dot blot Two-component signaling
This work was supported by NIH GM118894 and NSF CHE-1607532.