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.