Abstract
UV cross-linking assay is a standard method used to detect protein-RNA interaction. This method takes advantage of UV irradiation to trigger the formation of the covalently bonded RNP (ribonucleoprotein) complex that is more stable and makes it possible to be isolated in the denaturing conditions. Briefly, 32P-labeled RNA probe and proteins are incubated to form RNP complexes spontaneously; the mixture is then exposed to UV irradiation, followed by treatment with ribonuclease to remove RNA fragments not covalently bound to protein. The oligoribonucleotide-protein complexes are analyzed by SDS-PAGE, and the signals visualized by phosphorimaging. Competitive UV cross-linking assay is a method to determine the protein binding sites and specificity on the RNA substrate. In this assay, the excessive amounts of unlabeled competitor RNA are pre-incubated with the proteins prior to the addition of 32P-labeled RNA probe. If the competitor RNA comprises the protein binding region, the bindings between proteins and RNA probes will be competed and the radioactive binding signals will be reduced.
Keywords: UV cross-linking assay, Competition assay, Protein-RNA interaction
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from our previous publication: Huang et al. (2012). This research was supported by the Ministry of Science and Technology, Taiwan (grants NSC-98-2628-B-005-020-MY3 and NSC-98-2811-B-005-030).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.