I am trying to do an EMSA with unlabeled RNA an some proteins. RNA is about 700nt long in-vitro transcribed and the 2 protein are about 30 and 60 kDa with PIs 6-6.5. One of the proteins is the common fusion protein GST. After incubation of about 150ng of RNA with about 1ug of protein i add RNA loading dye and load half of it into a native 5% TBE PAGE gel and run it in 1x TBE . The RNA alone migrates just fine but whenever i add a decent amount of protein with the sample, I don't detect anything from those lanes with SYBR Gold staining.
Can anyone help me figure out why even the RNA from the non-interacting RNA+protein mixtures are not migrating into the gel?
Perhaps there is a trace of RNase in the protein sample, causing the RNA to be degraded. Try including some RNasin (RNase inhibitor protein).
Adam B Shapiro my proteins are recombinant purified proteins from E.coli. Should i aim for an RNAse inhibitor that targets prokaryotic RNAses I and H as well(suspecting RNAse is from prokaryotic source), or would i be fine with the common RNAse A, B, and C inhibitors such as RNAsin(suspecting RNAse is from external contamination) ?
Mix the RNA sample with the appropriate loading buffer. If running a denaturing gel, add equal volumes RNA sample and 2× denaturing loading buffer. If running a native gel, add 1 volume of 5× nondenaturing loading buffer to 4 volumes of RNA sample. Heat the samples for the denaturing gel at 94 °C for 5 min. https://www.sciencedirect.com/science/article/pii/B9780124200371000166
Kindly check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687016/
Hi Vicken,
My suggestion to use Hydrogen peroxide agarose gel for better RNA+protein mixtures migration.
Hope this may help you.
Have a look at this:
https://www.thermofisher.com/us/en/home/references/ambion-tech-support/nuclease-enzymes/tech-notes/the-right-choice-for-protecting-your-rna.html#:~:text=RNase%20inhibitors%20are%20typically%20used,several%20common%2C%20but%20diverse%20sources.&text=It%20is%20used%20in%20large,used%20in%20ribonuclease%20protection%20assays.
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