Hi,
I am running an assay that requires the disassociation of a small molecule from an albumin binding protein. The disassociation should occur in the presence of antibodies, so it can't be too denaturing.
Thanks for any recommendations.
If the affinity of the small molecule is not too high, it may be possible to remove it simply by passing the protein through a gel filtration column. This should work for micromolar dissociation constants, but probably not for nanomolar ones.
It may help to use mildly chaotropic conditions, such as 1 M NaCl, although this risks causing some protein aggregation.
Precipitating the protein with ammonium sulfate a few times can be helpful in removing tightly bound ligands.
If the ligand relies on a divalent cation to bind (e.g. Mg2+), adding EDTA to the buffer can be helpful.
Thank you for taking the time to respond to my question. I cannot pre-process the protein necessarily. I need to add something to the assay reagent that will cause the molecule to be released from the binding protein so that antibody can detect the molecule.
- Badges
- Science topic