In vitro RNA-protein Binding Assay by UV Crosslinking   

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Original research article

A brief version of this protocol appeared in:
Mar 2012


Because covalent bond can form between RNA and its binding proteins after UV irradiation, UV cross-linking is widely used to identify the specific RNA binding proteins. This protocol is described in details as follows.

Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Zhang, H. and Zhou, M. (2012). In vitro RNA-protein Binding Assay by UV Crosslinking. Bio-protocol 2(21): e287. DOI: 10.21769/BioProtoc.287.

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Guosheng Qu
1. I notice that ATP is added in the RNA-protein binding mixture before UV-crosslinking. Is it necessary for UV-crosslinking? If yes, why?

2. I did iCLIP by mixing RNA with protein in a different buffer (50 mM Tris-HCl, pH 6.8; 140 mM NaCl; 0.05% Triton-X100) and UV-cross-linking with 400 mJ/cm2. Under this condition, IP works and UV caused RNA damage is seen but no UV-crosslinks between RNA and protein have been detected (I use reverse transcription to detect peptidyl ligands left on the RNA after proteinase K digestion). Do you think what my troubles could be?
1/21/2016 7:08:48 AM Reply