期刊
RNA
卷 17, 期 1, 页码 14-20出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.2428111
关键词
fluorescence polarization; EMSA; gel shift; fluorophore; post-synthetic labeling; affinity; protein-RNA interaction
资金
- National Institutes of Health [GM081422]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM081422] Funding Source: NIH RePORTER
Sequence-specific recognition of nucleic acids by proteins is required for nearly every aspect of gene expression. Quantitative binding experiments are a useful tool to measure the ability of a protein to distinguish between multiple sequences. Here, we describe the use of fluorophore-labeled oligonucleotide probes to quantitatively monitor protein/nucleic acid interactions. We review two complementary experimental methods, fluorescence polarization and fluorescence electrophoretic mobility shift assays, that enable the quantitative measurement of binding affinity. We also present two strategies for post-synthetic end-labeling of DNA or RNA oligonucleotides with fluorescent dyes. The approaches discussed here are efficient and sensitive, providing a safe and accessible alternative to the more commonly used radio-isotopic methods.
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