I want to isolate total RNA and protein from the same sample. Isolated proteins should be in native form so that I can use it for further enzyme activity studies. Can someone please suggest any methods?
If you are working with cell culture, you can use this kit from Qiagen http://www.qiagen.com/Products/Catalog/Sample-Technologies/RNA-Sample-Technologies/DNA-RNA-Protein/AllPrep-RNAProtein-Kit#orderinginformation
I highly recommend it.
It's supposed that TRIzol reagent can extract RNA, DNA and Protein, but I never tried.
@alessandra. As fer as i know trizole reagent contains phenole & guanidine thiocyanate which are known to denature protein. Those proteins can be used for western blotting very well but I doubt whether we will get active protein for enzyme assay.
If you want to isolate RNA-protein duplex, you could probably use biotinylated probes. I found this might be helpful as well: http://www.invitrogen.com/site/us/en/home/References/Ambion-Tech-Support/rna-isolation/tech-notes/a-better-way-to-isolate-rna--and-protein-from-the-same-sample.html
Trizol will denature the proteins. I would perform a gentle lysis in a small volume of cold PBS with detergent, (NP-40 TritonX100 depending on your cells) and then I would divide the sample in two, one half for the protein assays and the other half for RNA extraction using standard methods (trizol, phenol etc.) . Add an excess of reagent to the RNA sample so that you don't dilute the denaturing reagent too much.
I think you would need to be very careful if you are going to try and keep enzyme activity of your proteins. At least part of the reason Trizol or other reagents are used is specifically to denature RNAses, which are very efficient at breaking down RNA. If you use conditions that keep the enzymes intact, you may lose RNA quite dramatically. Personally, I would advise my students not to try and get native protein and intact RNA from the same samples. However, if you want to try, use a cocktail of protease inhibitors (you can buy them) and some divalent cation chelators to inhibit enzymes while you are doing the isolation, then use column chromatography or dialyssi to change the medium. Good luck
The quality and yield of protein I have had from protein extraction with Trizol has never been satisfactory for my needs. If you do use Trizol make sure you lyse plenty of cells; it may over-come the yield issue but as for quality I'm not sure. I used protein for western blotting so it was going to get denatured anyway. If as others have suggested protein is denatured by Trizol extraction, a kit would be the way forward,
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