title: Separation and Detection of Phosphorylated and Nonphosphorylated BvgA, aBordetella pertussisResponse Regulator,in vivoandin vitro.
abstract: Protein phosphorylation plays a central role in signal transduction in bacteria. However, separation and detection of the phosphorylated protein from its nonphosphorylated form remain challenging. Here we describe a method to detect phosphorylation of theBordetella pertussisresponse regulator BvgA, which is phosphorylated at an aspartate residue (Boulangeret al., 2013). This method is based on the proprietary adduct, Phos-tag™, a dinuclear metal complex, which together with Zn2+or Mn2+, forms a complex with a phosphomonoesterdianion, such as the phosphorylated aspartate of a response regulator (Barbieri and Stock, 2008; Kinoshita and Kinoshita-Kikuta, 2011). Forin vivodetection,B. pertussiscells are lysed in mild formic acid at 4 °C to minimize the disruption of the phospho-aspartate bond, and the phosphorylated BvgA is separated from its nonphosphorylated form by electrophoresis (SDS-PAGE) containing Phos-tag™. Both forms of BvgA are subsequently detected by Western Blot analysis. Quantification of the level of phosphorylated BvgA formed after treatment with acetyl phosphatein vitrois also easily accomplished. Thus, this technique allows one to readily assess the levels of BvgA phosphorylation inB. pertussisand inE. coliunder different laboratory conditionsin vivoor after phosphorylation under varying reaction conditionsin vitro(this research was supported in part by the Intramural Research Program of the NIH, NIDDK).
authors.affiliation: Center For Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA.
authors.forename: Qing
authors.initials: Q
authors.lastname: Chen
authors.affiliation: NIDDK, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Alice
authors.initials: A
authors.lastname: Boulanger
authors.affiliation: NIDDK, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Hinton
authors.affiliation: NIDDK, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Stibitz