CLIP: UV cross-linking and immunoprecipitation technique. See the papers by Robert B. Darnell at Rockefeller University: http://www.rockefeller.edu/labheads/darnellr/CLIP.php

CLIP is an in vivo technique that works well not only from tissue culture cells, but from tissue as well.

A simple IP and protein purification will pull down the entire protein-RNA complex, and you will not be able to discriminate between direct and indirect interaction.

Hi Jinqiang,

1) Recombinant form of your protein binds to your RNA sequence of interest. That means no other potential bridging factors are around. You could do RNA gelshifts or UV crossinking of protein to radilabelled RNA or "Northwestern"

2) More in vivo like: UV crosslinking of extracts containing your radiolabelled RNA. Run a protein gel, blot and identify by first autoradiographie and then with your protein specific antibody. If your lucky both signal match.

Sure there are more approaches in the RNA world

good luck

Surface plasmon resonance is always popular. There is also isothermal calorimetry (http://onlinelibrary.wiley.com/doi/10.1002/bip.20816/full). You could do a fluorescence quenching assay on a fluorimeter with recombinant protein as well.

Hi Daniel,

your suggestion is very interesting (SPR = Biacore?). A collegue of mine (phD student) wants to do that. Do you know any reference?

would be great

thanks

Michael

AFM technique it is also quite popular if you want check I suggesting you to check this article and see if you can find what you need:

ITC and SPR are the methods of choice

make IP for your protein using specific antibodies, then after elution, denature the proteins with an RNA friendly solution, then convert the mRNA to cDNA by reverse transcription. if you know your RNA sequence, then design primers to amplify the cDNA...that should proof the existence of the interaction in the first point..

this only my idea, it may be stupid or may work

Michael,

Yes, the commercial version of SPR is biacore. Here is a reference describing its use for the determination of RNA-protein interaction kinetics.

http://www.sciencedirect.com/science/article/pii/S1046202302000129

on SPR here is another latest paper on nucleic acid and protein interaction

http://www.ncbi.nlm.nih.gov/pubmed/23673861

Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4.

Blood. 2013 Jul 11;122(2):272-81. doi: 10.1182/blood-2013-01-478966. Epub 2013 May 14.

CLIP: UV cross-linking and immunoprecipitation technique. See the papers by Robert B. Darnell at Rockefeller University: http://www.rockefeller.edu/labheads/darnellr/CLIP.php

CLIP is an in vivo technique that works well not only from tissue culture cells, but from tissue as well.

A simple IP and protein purification will pull down the entire protein-RNA complex, and you will not be able to discriminate between direct and indirect interaction.

Monitoring interactions in solution under near-physiological conditions can be done by X-ray scattering or SAXS under many different conditions with tiny volumes and 1-2ug/ul concentrations at the level of 96-well plates (Nature Methods 6:606-12, 2009). Complexes scatter as a function of the square of the number of electrons in the components so SAXS is very sensitive to detecting interactions and complex formation. One can directly determine mass from the scattering experiment without knowing the protein concentration and without requiring a fully ordered protein (Nature 496, 477-81, 2013 ) SAXS can be done at low costs by Mail-In as part of NIH-funded efforts see http://bl1231.als.lbl.gov and S. Classen et al., J Appl Crystallogr. 46,1-13. So you could test an RNA-protein interaction under multiple conditions simply by sending a 96-well plate for SAXS analyses.

Some RNAs have a binding tendency towards certain matrix like DEAE sepharose whereas the corresponding proteins might not. In such a situation if your proteins and RNA are known to interact, they will only bind to it when RNA was preloaded onto matrix and are seen in elutes. Without the presence of bound RNA in the matrix, the proteins will pass in flow through. (See Krishne Gowda et al.)

Thanks for all your suggestions, I will read the mentioned references and hopefully find a way to demonstrate how my target RNA-proteins complex bind together.

Thanks again.

Addition to the methods already suggested, you can try fluorescence thermal shift assay using a qPCR machine and Sypro-Orange dye. Of course, isothermal calorimetry is also a good way. Both of these methods, however, require purified protein.

You should have a look at MST (microscale thermophoresis) its a Novel method for measuring binding kinetics in solution