We have purchased recombinant protein in the form of dimers, trimers and oligomers (confirmed by carrying out non reducing gels). In order for successful ligand binding assays we anticipate that monomeric proteins are essential to determine if the ligand binds sufficiently to the protein receptor.
My question is twofold, does any one have experience breaking up dimeric proteins (with detergents or otherwise) for use in bioassays or expertise in ligand binding assay protocols?
Any thoughts or comments would be greatly appreciated!
Means a lot playing with salt and detergents and keeping in mind that the monomers will recombine as soon as the conditions are changed back to PBS etc.
More promising might be engineered proteins that cannot multimerize or adding synthetic peptides which mask the relevant sites.
There is the possibility that the oligomers bind to the ligand as well as the monomer, in which case it may not be necessary to find a way to dissociate them.
That depends on the bonds between the oligomers. Are they covalent? In that case, no matter how much detergent or salt you add, they will stay together. If they are bound by cystein bridges, you can add reducing agents.
If they are bound non-covalently, then it depends, whether it's electrostatic (in that case a salt will help) or hydrophobic (in that case you need detergent).
As Wolfgang wrote, this will require a little of playing. And it may be better to use a mutated version, unless you specifically require the one, which is able to bind.
Regarding the binding assay, what is used in our area is replacement of radioactive compound, where you have one standard radioactive compound and then several other compounds, which you add in excess and then determine how much radioactivity stayed bound. This can be done by immobilization, e.g. fusion with gluathione S-transferase, streptavidin or FLAG-tag and pulling your protein down and measuring remaining radioactivity.
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