I want to assemble an RNA component that presumably fits within a protein (not on surface). If anybody come across any ideas or people who actually do this kind of stuff. Please let me know a way.
Do you have a reason to suspect that it cannot assemble on its own when the components are mixed? How do you think the complex assembles in vivo - do you need another factor? I would try a simple protocol first - refold the RNA (heat and either snap cool or slow cool in buffer +/- monovalent, then add divalent metals and bring to reaction temp), then add the protein and see if the complex forms. You may have to try a few different salt concentrations and pH values. If it doesn't form, then worry about a more complicated protocol.
Hi,
- First do you know how your protein binds to the RNA? Does it have RNA binding domain that recognizes a specific sequence or just bind to the secondary structure of RNA? This would be important to decide whether you need to heat your RNA sample first or not. You might also need to check if your RNA contains the specific target of your protein.
- Just try incubation in binding buffer. Most RNA binding proteins would bind in this condition to the target RNA. Many protocols are available online. You can try this link for an example which I know works for my experiment http://www.bio-protocol.org/e287
- If it is necessary you could crosslink your RNA-protein complex by irradiate your mixture under UV crosslinker. This will help to freeze your interaction to survive some extreme buffer conditions, i.e during high salt washing. This especially important when your complex is weakly interact or temporary.
Good luck!
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