Hi Behdad, are you just interested in proteins that bind RNA?

If so, you could immobilize RNA onto a resin and pass the cytoplasmic proteins through it. If you are just looking for proteins that bind generic RNA (and not specific RNA), all commercial RNA preparations could be used. The proteins could be eluted using urea or RNAse.

One problem though is dealing with cytoplasmic proteins of lysed cells. I used a method of lysing the cells in detergent and shearing the DNA/RNA in the prep by repeatedly pulling and expelling the lysates through a small bore needle.

Thanks. What we'd like to do is to take cellular protein extracts (e.g. cytoplasmic or nuclear) and pull down all protein-bound RNA. I'm guessing it'll take a CLIP sort of experiment with X-linking of RNA to proteins then protein pull-down, but we don't want to bias the protein pull-down by using Ab IP. Need something generic that would work even with RNA-X-linked proteins. Any ideas?

Yeah, but X-linking RNA to proteins is tricky since there is no way to ensure specific crosslinking between the two without introducing unique functional group(s) on the RNA.

Generic crosslinking methods will just give you a ball of snot.

If you are not adverse to working with radiolabels, you could try incubating the cells with 3H or14C uridine. Harvest the cells in the presence of RNAse and run them on a gel. Native PAGE will not dislodge tightly bound protein ligands. See how many bands light up.

We're hoping to use the RNA for sequencing later. This option might introduce downstream problems.