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 language | English (US) |
---|---|
Title of host publication | RNA Remodeling Proteins |
Subtitle of host publication | Methods and Protocols |
Publisher | Springer New York |
Pages | 369-383 |
Number of pages | 15 |
ISBN (Electronic) | 9781493922147 |
ISBN (Print) | 9781493922130 |
DOIs | |
State | Published - 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)