Dear all,
Interest in RNA-protein interactions is booming. In the protocols of CLIP and RIP, UV radiation is used to cross-link RNA binding proteins to the RNA that they are bound to. However, the protocols do not mention how long for UV cross-linking.
I would be really appreciated if anyone could suggest detailed conditions, especially the UV cross-linking time and energy.
Hui
The time and level of UV exposure varies from case to case. You need to do a "time titration" and a "UV strength titration" for your own experimental case. That is, do several set-ups and vary the times (e.g., 5, 10, 15, 20, 25 minutes, etc.), while keeping the UV strength constant. Then do another set of setups where you vary the UV strength, this time keeping the time (ideal time determined previously) constant. This is called "optimization" of your experiment. Danke schoen und tschuss.
I only know the crosslink conditions used in iCLIP experiment. I guess it should work the same. For crosslinking you would have to use a stratalinker device (or similar) and irradiate the cells (10 cm dish in 6 ml PBS) with 150 mJ/cm2 at 254 nm. It's not the time that matters but the absolute amount of energy irradiating the cells. These conditions generate robust crosslinks.
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