- December 31, 2015 at 7:14 am #18206kishselParticipant
I m currently working on Proteus mirabilis HFQ protein binding sRNA identification and characterization. I have constructed the recombinant plasmid by ligating HFQ gene from Proteus mirabilis into pET 28 recombinant vector and subsequently overexpressed the HFQ protein using IPTG induction in a competetnt BL-21 E.coli cell.. This protein was further purified by elution with an appropriate concentration of imidazole.. I have also extracted the total RNA from Proteus mirabilis using Trizol procedure.
I am ready for the Protein-RNA binding assay, but i need help in a standardized procedure to execute the Protein-RNA binding assay.
- December 31, 2015 at 4:21 pm #115867claudepaParticipant
Hi. I am sure you will have a precise answer from a researcher using this technic. I am only curious about sRNA. I know many RNAs but not this one. I suppose that the principle of the technic after binding is the digestion of unprotected RNA by RNase. Then digestion of the protein by a protease and finally analysis of the protected RNA ? Have a good work.
- January 2, 2016 at 12:36 pm #115870kishselParticipant
sRNA are bacterial small non-coding RNAs. My objective is in the identification and characterization of Proteus mirabilis HFQ protein binding small non-coding RNAs.
- January 2, 2016 at 7:55 pm #115871claudepaParticipant
Thank you. I am more focused on eukaryotic cells, where there are many regulatory small RNAs. Good to know that there are also non coding RNAs in bacterias. I know that RNase protection assay can be used to identify RNA sequences for RNA binding proteins.
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