I am trying to run a positive control RNA EMSA with a known RBP with TYE665-labelled dsRNA, following the same protocol as was previously published. Despite including RNase inhibitor in the reaction, it appears as though the RNA signal decreases drastically at the lowest protein concentration, compared to unbound probe, without a reciprocal gain in signal at a higher molecular weight. At high protein concentration, I'm starting to see what looks like a shift, however the overall signal intensity is quite low.
Has anyone seen this before/ Does anyone have any troubleshooting suggestions for this?
Thank you!
Attached image is an example. 10ul reaction containing 10nM dsRNA and increasing [protein], 2ul loaded in duplicate, with unbound probe as the first 2 lanes on the left. 10% Native PAGE after 10 min of polymerization.
ds RNA are supposed to be quite resistant to RNAse (exept if it is only partially ds) how did you label the probe? radiactivity at the 5' ends? it seems also that you have increasing quantity of "material" in the wells and smears: can you quantify the signal in the whole lane? sometimes the nucleic-)acid protein can dissociate when running the gel (too hot (run the gel in the cold room), not the right buffer (try 0.5 TAE) weak interaction... ) resulting in a smear. protein-RNA interaction coud give high molecular complexes that do not penetrate into the wells
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