I am carrying out the iCLIP protocol and I am having problems with a lot of background in my high RNase control. Instead of seeing a sharp band where at the correct molecular weight of my protein, I see instead a black smear all throughout the lane. Darker than that of my low RNase control. Has anyone had anyproblems with RNase digestion followed by gamma ATP 5' RNA labelling? The steps I have followed are outline below and as always any suggestions would be very helpful...Thanks!
Crosslink RNA interacting with Protein via UV irradiation.
Lyse cells and treat with RNase I (Ambion) at a high (1/50) and low (1/500) dilution for 3 minutes at 37C
IP O/N with stringent conditions
All the following steps are carried out on bead:
Treat with Alkaline Phosphatase and then ligate 3'adaptor
Next the RNA is radiolabelled at its 5' end with gamma ATP via PNK (3 prime minus NEB)
The protein complexes with their heterogenous length RNAs are separated out on a 3-8% Tris Acetate gel.
Next I can visualise the presence of RNA with an Xray film. However, the higher concentration of RNase I only see a very heavy black smear. The high RNase control is used to check that the IP has been specific because a higher concentration of RNase will lead to shorter RNA allowing the protein to migrate normally and therefore a sharp band should be present at the size of the protein I am immunoprecipitating.