Fluorescence reporters for Hfq oligomerization and RNA annealing.
Methods Mol Biol
; 1259: 369-83, 2015.
Article
em En
| MEDLINE
| ID: mdl-25579597
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.
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1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Escherichia coli
/
Fator Proteico 1 do Hospedeiro
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Polarização de Fluorescência
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article