I want to identify the host proteins that interact with the non-coding RNA of virus. Is it possible to do the screening on the similar lines as done in protein-protein interactions using Y2H.
RNA pulldown followed by mass spectrometry analysis.
You could bind your RNA of interest to beads, and then incubate your mix with a cellular lysate. After washing, you should fish the proteins bound to your RNA and identify them by MS.
Depending on the length of your RNA of interest, you could prepare it either by in vitro transcription (for long RNA) or buy it synthetized (provided by IDT as example). In my case, I did it for small ncRNA. I bought my sequence (with IDT) with a biotin as modification and I fixed it on streptavidin-beads. It worked pretty well.
But don't forget to use a control (beads alone and/or beads with an unrelated RNA) then you can discriminate between the proteins specific of your RNA and the proteins bound to the beads. It will be a real help to limit the number of candidates for validation as the beads alone are binding a lot of proteins.
RNA pulldown followed by mass spectrometry analysis.
You could bind your RNA of interest to beads, and then incubate your mix with a cellular lysate. After washing, you should fish the proteins bound to your RNA and identify them by MS.
Depending on the length of your RNA of interest, you could prepare it either by in vitro transcription (for long RNA) or buy it synthetized (provided by IDT as example). In my case, I did it for small ncRNA. I bought my sequence (with IDT) with a biotin as modification and I fixed it on streptavidin-beads. It worked pretty well.
But don't forget to use a control (beads alone and/or beads with an unrelated RNA) then you can discriminate between the proteins specific of your RNA and the proteins bound to the beads. It will be a real help to limit the number of candidates for validation as the beads alone are binding a lot of proteins.
Dr. Haas thanks for your reply. I would like to know that whether 2DGE is must for MS. Wel, if its so then its not possible in my lab. Can you plz suggest me some other alternatives to screen host proteins binding to a known RNA.
Thanks
I would recommend nanoLC (liquid chromatography)-MS/MS analysis. Check in your institutes or the neighborig institutes if one of them have these facilities. I'm not doing the identification myself but a MS platform was doing it for me. Otherwise, if you have putative candidates picked from the litterature, you can run western blot and see if these candidates are pulldown or not.
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