If your protein forms a stable complex with RNA and you have an Acta system that can measure two wavelengths at the same time, then you can also try the following

Purify your protein and make sure you remove all traces of RNA from it. You can achieve this by using RNAse A, ion exchange chromatography or, in difficult cases, you can use high ionic strength washes (I have a protocol for this if you need it).

A) Run a sample of the RNA-free protein on an analytical size exclusion chromatography (SEC) column (you can also use a purification SEC column, but you'll need larger volumes of protein). Measure the absorbance at 280 nm and 260 nm. If your protein is pure and RNA-free you'll get a single peak where the Abs 280 nm trace is larger than the 260 nm trace.

B) Now run a sample of your RNA on the column (the fragments should be homogeneous, the same length and sequence). This will give you a single peak with a large Abs 260 nm trace and a very low Abs 280 nm trace. The peak should elute at a different position from the protein peak because the RNA fragments are a different size from the protein (usually).

C) Lastly, mix the RNA-free protein and the RNA and let them interact for at least 30 min to 1 hour before you run the mixture on the column. If they don't interact, you'll get a trace with two peaks that look like the ones in A) and B). If they do interact, the protein peak will have a larger Abs 260 nm trace than 280 nm. Assuming you added excess RNA to the mix, then you should also get an RNA peak, but the overall size of this RNA peak should be smaller compared to B), as some of the RNA is now in the protein peak instead.

Now you can also do a very crude assay on an agarose gel to directly visualise the RNA. Using an RNA ladder, run a sample of your RNA-free protein peak from A), a sample of your RNA peak from B) and a sample of your protein peak from C). The protein in the samples will stay in the wells and all nucleic acids will run down the gel. I find that you simply need to add agarose gel loading dye to the protein samples (the kind you use for DNA or RNA samples) and they're ready to be loaded. In the past I also boiled the protein samples first to denature them and make sure they release the RNA before adding the loading dye, but this doesn't seem to be necessary.

If your initial RNA-removal step worked, you shouldn't see an RNA band in A). If you see one in C), then they definitely interact.

You can also run an extra sample on the agarose gel, of the protein that hasn't been treated to remove the RNA. This way you can see if the protein binds RNA from the expression system (e.g. transgenic proteins expressed in E. coli can bind E. coli RNA, which happened to me in several instances).

You can use RNA electrophoresis mobility shift assay, RNase protection assay. These are the two simple methods that can be used to detect RNA-protein interaction.

Mobility Shift (EMSA assay) is indeed a very simple and efficient method. You can try also a mass-spectrometry – in many cases complexes are sufficiently stable and can be detected in MALDI or ESI-MS.

Thank you. 

For the EMSA you need to know the nucleic acid that you add into the mixture with protein to see if it binds some protein from the lysate.

In my case I know the protein's in the lysate and I know what it is, I need to know if there are any small RNAs that can bind my protein.. basically if I can "fish-out" these RNAs out of my lysate by doing the IP.

What I planned to do after IP is to Trizol the IP'ed protein-RNA mixture, isolate small RNA and sequence them (by NGS for ex.). 

For your purpose, perhaps the old 'northwestern' blotting method might be of use.  Of course, the immobilized protein should be in some sort of 'native' configuration and the putative RNAs to probe with in their correctly folded state…  Maybe people used to fishing out microRNAs could have useful tricks?

you can try to tag your protein with  a flag or HA or GFP, then you IP the tagged protein instead. there are many commercial Abs against these small proteins that are excellent for IP.

you may purify the protein, order some DNA and use Microscale Thermophoresis to determine the Kd and stochiometry. Easy, simple, very fast and you dont need much protein....

I suggest you consider using X-ray scattering (SAXS). As RNA scatters more strongly that protein it is possible to separately assign protein and RNA components. A SAXS experiment at a synchrotron, which is accessible through user proposals, takes ~ 1 second/sample for data collection plus time for sample exchange so many hundreds of samples or conditions can be examples per day - see RNA. 2010 Mar;16(3):638-46. doi: 10.1261/rna.1946310 and Annu Rev Biophys. 2013;42:415-41. doi: 10.1146/annurev-biophys-083012-130301. You might be able to examine the X-ray scattering of your protein by itself and then in the presence of RNA. The formation of a complex would be detected robustly as complexes scatter with the square of the number of electrons so the complex would show up strongly. 

Mark,

If you do have your protein, why not to bind it directly to activated gel or magnetic particles and do pull down w/o antibodies? 

Thank you colleagues.

Krzysztof, I have a protein in lysate, so it's not homogene and overexpressed.. Or what do you mean by binding my protein directly to magnetic particles?! I've never heard about that technique! Sounds very interesting! Can you spare some details, please?

Thanks!

Mark, 

I misread your post and was sure you have your protein pure. That is required for approach I was proposing. If you do have pure protein you can bind it covalently to any surface activated with amine, sulfhydryl or carboxyl/hydroxyl groups (here is link to wonderful book describing how: http://www.amazon.com/Bioconjugate-Techniques-Third-Edition-Hermanson/dp/0123822394).  If you buy magnetic particles with for example amine groups introduced to the surface, you can bind your protein to them using proper reagent (http://www.piercenet.com/method/chemistry-crosslinking#nhsester). This way you will have magnetic particles with your protein on the surface. Next you can fish out from lysate whatever binds to your protein by suspending magnetic microsphres in it, rotating for some time and next collecting microparticles using magnetic stand. Of course it will need a lot of effort to eliminate unspecific binding, wash out all not bound  stuff and use proper controls, so it may be effort-consuming, but in my opinion worth trying. Alas, you do not have your protein pure or expressed.

In your situation maybe a kind of co-purification may work? For example gel filtration and identification of fraction with your protein and checking if any RNA coeluted with it? Also will need proper care in designing. 

Question, If commercial antibodies do not bind your protein, how you detect it specifically? Is it a kind of enzyme with highly specific substrate?

another thought:

if you do know sequence of your protein or conserved sequence in the family it belongs, maybe you can send it to company like this: (http://www.genscript.com/custom-antibody-production-services.html?src=pullmenu). Next you can use these antibodies instead commercial you re using now.

I did that once and they produced very good antibodies for my protein. But there is no guarantee it will work for sure.

If your protein forms a stable complex with RNA and you have an Acta system that can measure two wavelengths at the same time, then you can also try the following

Purify your protein and make sure you remove all traces of RNA from it. You can achieve this by using RNAse A, ion exchange chromatography or, in difficult cases, you can use high ionic strength washes (I have a protocol for this if you need it).

A) Run a sample of the RNA-free protein on an analytical size exclusion chromatography (SEC) column (you can also use a purification SEC column, but you'll need larger volumes of protein). Measure the absorbance at 280 nm and 260 nm. If your protein is pure and RNA-free you'll get a single peak where the Abs 280 nm trace is larger than the 260 nm trace.

B) Now run a sample of your RNA on the column (the fragments should be homogeneous, the same length and sequence). This will give you a single peak with a large Abs 260 nm trace and a very low Abs 280 nm trace. The peak should elute at a different position from the protein peak because the RNA fragments are a different size from the protein (usually).

C) Lastly, mix the RNA-free protein and the RNA and let them interact for at least 30 min to 1 hour before you run the mixture on the column. If they don't interact, you'll get a trace with two peaks that look like the ones in A) and B). If they do interact, the protein peak will have a larger Abs 260 nm trace than 280 nm. Assuming you added excess RNA to the mix, then you should also get an RNA peak, but the overall size of this RNA peak should be smaller compared to B), as some of the RNA is now in the protein peak instead.

Now you can also do a very crude assay on an agarose gel to directly visualise the RNA. Using an RNA ladder, run a sample of your RNA-free protein peak from A), a sample of your RNA peak from B) and a sample of your protein peak from C). The protein in the samples will stay in the wells and all nucleic acids will run down the gel. I find that you simply need to add agarose gel loading dye to the protein samples (the kind you use for DNA or RNA samples) and they're ready to be loaded. In the past I also boiled the protein samples first to denature them and make sure they release the RNA before adding the loading dye, but this doesn't seem to be necessary.

If your initial RNA-removal step worked, you shouldn't see an RNA band in A). If you see one in C), then they definitely interact.

You can also run an extra sample on the agarose gel, of the protein that hasn't been treated to remove the RNA. This way you can see if the protein binds RNA from the expression system (e.g. transgenic proteins expressed in E. coli can bind E. coli RNA, which happened to me in several instances).

Mark: to get around the antibodies that don't work well, you could transfect your cells with the tagged version of your protein using a tag that has robust antibodies to use for IP (FLAG, HA etc).

One way is to used is to used yeast-3-hybrid. (PMID: 15613539) The strategy involve expressing your protein fused to Gal4 AD domain and your RNA fused to MS2 hairpin in yeast vectors.

Another option is to anchor your RNA to beads and pull out the protein with the crosslinked beads and reveal protein with the antibody that only work on a WB (also the sample can be analyzed by mass spec to determine all binders). (PMID: 20876833)