Paraformaldehyde Crosslinking Co-ip Problem

[Anton Chigger]

For a couple of months now I've been working on a coimmunoprecipitation of a few membrane proteins that are involved in peptidoglycan synthesis and regulation in a certain gram positive bacteria. This procedure starts with a step in which I add anywhere from .1 to .4% paraformaldehyde to a PBS solution containing my cells. My yield has not been very good, and I've since found that I am losing roughly 70-80% of my desired protein during the crosslinking step, before anything else is done to the cells.

Does anyone here have an idea of what might be happening or ever heard of this problem before? I checked the supernatant after spinning down the cells and the missing protein is not there. My other idea was that the proteins are forming a large insoluble complex, but I frankly have no idea how I might solubilize something like that in this context. Thoughts?

[mmorgan]

Originally Posted by Anton Chigger

For a couple of months now I've been working on a coimmunoprecipitation of a few membrane proteins that are involved in peptidoglycan synthesis and regulation in a certain gram positive bacteria. This procedure starts with a step in which I add anywhere from .1 to .4% paraformaldehyde to a PBS solution containing my cells. My yield has not been very good, and I've since found that I am losing roughly 70-80% of my desired protein during the crosslinking step, before anything else is done to the cells.

Does anyone here have an idea of what might be happening or ever heard of this problem before? I checked the supernatant after spinning down the cells and the missing protein is not there. My other idea was that the proteins are forming a large insoluble complex, but I frankly have no idea how I might solubilize something like that in this context. Thoughts?

When you say you "lose" your protein, it must end up somewhere. Is it in the pellet after you make your membrane lysate???

Crosslinking could cause your protein to become insoluble, also it could mask the antibody binding site (I assume you are immunoprecipitating).

I don't know the details of your protocol, but I would start be testing all your fractions for presence of the protein by western blotting (preferably with more than one antibody in case crosslinking changes it's immunoreactivity).

If you determine that the protein is indeed becoming insoluble you could try to start overcoming that. Since you have crosslinked, you can be reasonably harsh in solubilizing the proteins. You could try SDS, Urea, DTT perhaps with sonication.

Another possibility would be to try alternative crosslinking agents in the hope that your complex does not become insoluble.