I have a soluble receptor and I am trying to develop the binding assay conditions using radiolebeled ligand. My problem is separating the bound from free radioligand. I've tried the precipitation method and it sort of works but I think I am loosing a lot of signal due to ligand dissociation during the precipitation time. I've also tried using dextran-coated charcoal but that is extremely not reproducible, in addition the charcoal doesn't completely remove the free radioligand and that severly effects the results.
Doe anyone know a better and quick method of separating free from bound radioligand for soluble receptors? Any references or advise would be appreciated,
Marek

You might consider using filter plates (we use Millipore filter plates) and a vacuum manifold. There is a good chance that you filter all unbound ligand.

kazey said:

You might consider using filter plates (we use Millipore filter plates) and a vacuum manifold. There is a good chance that you filter all unbound ligand.

thanks for your thought,
do you use one of those molecular cut-off ones?
I tried the Microcon YM-3 but it takes forever, maybe a larger pore size?

Refer to This Related Reply in the Protein Chemistry forum

I have GPCRs from transfected CHO for binding assays. The problem is that it no longer couples to G-protein or inhibits c-AMP, but the antibody says that the receptor is highly expressed on the cell surface.
What can be a problem with the GPCRs that explain it?
I would appreciate any help.

kazey said:

You might consider using filter plates (we use Millipore filter plates) and a vacuum manifold. There is a good chance that you filter all unbound ligand.

You can remove the unbound radioligand succeefully if you could work with PerkinElmer GF/B Filterplates and perkinelmer cell harvester.