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