Malmqvist, T., Spickett, C., Gallo, J.-M. and Anthony, K. (2017) A UV cross-linking method combined with infrared imaging to analyse RNA–protein interactions. Biology Methods & Protocols. 2(1), pp. 1-4. 2396-8923.
| Item Type: | Article |
|---|---|
| Abstract: | Photo cross-linking of proteins with short RNA oligomers is a classical method to study RNA–protein interactions that are implicated in many aspects of RNA metabolism and function. Most commonly, this involves the use of [c-32 P]-labeled RNA probes. Although very sensitive, these procedures are complicated by the safety issues associated with the use of radioiso-topes. Here, we describe a modified UV cross-linking method using oligonucleotide probes end labelled with the infrared dye IRDyeV R 800. After UV cross-linking, proteins are separated by SDS-PAGE and cross-linked products are visualized with the Odyssey V R Infrared Imaging system. This end labelling approach provides a streamlined alternative to random labelling which reduces the efficiency of in-vitro transcription. End labelling is also independent of the length of the probe, thus facili-tating quantitative comparisons. To validate the method, we have confirmed the binding of HuD to the 3 0 -UTR of the mRNA for the microtubule-associated protein tau, implicated in the pathogenesis of Alzheimer's disease. UV cross-linking of HuD with a labeled 21-mer probe was successfully performed using a recombinant purified glutathione-S-transferase–HuD fu-sion protein as well as with lysates from CHO cells transfected with HuD cDNA. UV cross-linking combined with infrared imaging offers a convenient and robust strategy to analyse RNA–protein interactions and their emerging importance in disease. |
| Uncontrolled Keywords: | RNA, RNA-binding protein, UV cross-linking, Odyssey, tau, HuD, neurodegeneration |
| Creators: | Malmqvist, Tony, Spickett, Carl, Gallo, Jean-Marc and Anthony, Karen |
| Faculties, Divisions and Institutes: |
Faculties > Faculty of Health & Society > Sports, Exercise & Life Sciences Research Centres > Centre for Physical Activity and Life Sciences |
| Date: | 5 June 2017 |
| Date Type: | Publication |
| Page Range: | pp. 1-4 |
| Journal or Publication Title: | Biology Methods & Protocols |
| Volume: | 2 |
| Number: | 1 |
| Number of Pages: | 4 |
| Language: | English |
| DOI: | https://doi.org/10.1093/biomethods/bpx009 |
| ISSN: | 2396-8923 |
| Status: | Published / Disseminated |
| Refereed: | Yes |
| URI: | http://nectar.northampton.ac.uk/id/eprint/11040 |
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