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Fluorescence Reporters for Hfq Oligomerization and RNA Annealing

  • Subrata Panja
  • Sarah A. WoodsonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

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.

Key words

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

Notes

Acknowledgements

The authors thank T. Soper, Y. Peng, and A. Santiago-Frangos for helpful discussion. This work was supported by a grant from the NIH R01 GM46686.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.T. C. Jenkins Department of BiophysicsJohns Hopkins UniversityBaltimoreUSA

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