Hi,
I am trying to detect complex formation in the proteins that I'm working with. Can anybody suggest a simple protocol for protein-protein crosslinking?

Thank you.

Hello,

Though I can't recall a precise citation, you can crosslink proteins with formaldehyde. This is the same sort of crosslinking that's done for CHIP assays (which also entail un-crosslinking)...those are all over the web, and should be easy to find. I do have a question or two about your complex...

Is this complex stable enough to be isolated through conventional means without crosslinking? You might want to devise a scheme to obtain your complex that way, and here's why: formaldehyde crosslinking will complex all sorts of proteins with your protein of interest...all they have to do is be proximal to your protein of interest to be crosslinked, and so you will likely end up w/ a bunch of false positives in your data set.

The only other way I can think of to covalently couple your proteins is w/ glutaraldehyde, but then you'll run into the same problems as above, and I'm not so sure the binding is reversible in that case.

I hope this helps.

- Jon
jonmike.reed@gmail.com

Do you really want only crosslinking, or you want just to create the stable complex of homo or heterodimers? To create such complex people use leuzine zipper, or different helixes, or c-terminal cysteines , or different types of multimerizing tag.

I have heard good thinigs from a colleague of mine who used.... I think it was DSP from Pierce Biotechnology. They have lots of x-linkers and are great about giving protocols.

Some of them are reversible (ie the x-linking comes apart upon reduction in 2-ME), which is nice because then you know where to expect all your proteins on gel.

Good Luck,

Mountainman