title: Fluorescence reporters for Hfq oligomerization and RNA annealing.
abstract: Fluorescence spectroscopy is a sensitive technique for detecting protein-protein, protein-RNA, and RNA-RNA interactions, requiring only nanomolar concentrations of labeled components. Fluorescence anisotropy provides information about the assembly of multi-subunit proteins, while molecular beacons provide a sensitive and quantitative reporter for base pairing between complementary RNAs. Here we present a detailed protocol for labeling Hfq protein with cyanine 3-maleimide and dansyl chloride to study the protein oligomerization and RNA binding by semi-native polyacrylamide gel electrophoresis (PAGE) and fluorescence anisotropy. We also present a detailed protocol for measuring the rate of annealing between a molecular beacon and a target RNA in the presence of Hfq using a stopped-flow spectrometer.
authors.affiliation: T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD, 21218, USA.
authors.forename: Subrata
authors.initials: S
authors.lastname: Panja
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Woodson
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D035001:Host Factor 1 Protein / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein