Fluorescence reporters for Hfq oligomerization and RNA annealing

Subrata Panja, Sarah A. Woodson

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationRNA Remodeling Proteins
Subtitle of host publicationMethods and Protocols
PublisherSpringer New York
Pages369-383
Number of pages15
ISBN (Electronic)9781493922147
ISBN (Print)9781493922130
DOIs
StatePublished - Jan 12 2015

Keywords

  • Fluorescence anisotropy
  • Hfq
  • Molecular beacon
  • Protein oligomerization
  • RNA chaperone
  • Stopped-flow fluorescence

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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