I'm working with a protein that might be having RNAse ativity. I would like to test this and get the constants if possible. Does anybody have a protocol for that? I found some in papers but I would like you to recomend me one that works fine! Thank you so much.
There are many ways to do this:
If your RNAse is specific, incubate the protein in question together with its target (the RNA), then run a gel of the RNA and see if it's still intact (one band) or cut (several bands or at least 2).
If your RNA is not specific, gest some large RNAs (ribosomal RNAs for example) and incubate together with the protein, then run a gel again.
A somewhat dirtier approach is to label some RNA with P-32, precipitate, the dissolve again. Then add your protein, incubate, and then precipitate again. If there is RNAse-activity, the portion of the total counts you can precipitate should become lower.
If you are looking for a method that doesn't involve running gels or using radioactively labeled RNA, consider using the fluorescent dye RiboGreen (Life Technologies), which has RNA-dependent fluorescence. You can use a fluorescence plate reader. RiboGreen will also detect DNA, so the method works best if the RNA is free of DNA.
Thank you Andreas and Adam!
I did run the gel, since I didn't have to wait for it. It seems to work!
Thank you so much!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins