title: Detection of histidine kinases via a filter-based assay and reverse-phase thin-layer chromatographic phosphoamino acid analysis.
abstract: The methods that detect histidine phosphorylation have largely been either laborious or difficult to apply quantitatively. The major difficulty in assessing for its presence is its alkali-stable, acid-labile nature. While an assay that detects alkali-stable phosphorylation has been developed, it does not distinguish phosphohistidine from other alkali-stable phosphoamino acids. Using this established method, we extend the assay to facilitate the specific detection of phosphohistidine. We use the acid-lability of phosphohistidine as a defining feature in our approach for its detection. In addition, reverse-phase thin-layer chromatography was utilized to conclusively demonstrate the viability of the conditions that we implement in the assay for the selective detection of phosphohistidine. In summary, this report describes a rapid filter-based kinase assay that quantitatively measures histidine kinase activity, even in the presence of tyrosine kinase activity.
authors: Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6100, Australia.
authors: Eiling
authors: E
authors: Tan
authors: Xin
authors: X
authors: Lin Zu
authors: George C
authors: GC
authors: Yeoh
authors: Paul G
authors: PG
authors: Besant
authors: Paul V
authors: PV
authors: Attwood
mesh_terms: D002855:Chromatography, Thin Layer / Q000379:methods*; D005656:Fungal Proteins / Q000032:analysis; D006639:Histidine / Q000031:analogs & derivatives* / Q000032:analysis; D000071677:Histidine Kinase; D006657:Histones / Q000032:analysis; D008567:Membranes, Artificial; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D006657:Histones; D008567:Membranes, Artificial; D006639:Histidine; D011494:Protein Kinases; D000071677:Histidine Kinase; C005436:phosphohistidine