RNase digestion followed by Radiolabelling iCLIP/CLIP - (Oct/17/2013 )

Hello People,

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!

Steps:

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.

a few thoughts...

how long do you expose the film? 32P is very energetic and may give you an apparent blob where the actual band may be sharp.

shorter exposures and/or smaller loadings may reduce the "blob".

on the other hand, you may be seeing a blob because of variable binding of the rna or proteolytic cleavage of the protein or variable cleavage of the rna.

the gel may be the cause. your sample may have flowed down the outside of the gel if there were places where the gel didn't adhere to the plate (bubbles against the glass). it may not have polymerized properly. the sample may not have stacked well (not normally a problem with a gradient gel but may still be a problem if the sample volume is very high).