Hi Raheleh,

There are two major considerations you should keep in mind in order to avoid introducing error into the assay: The time to equilibrium of the binding reaction and ligand depletion that occurs during the binding assay. That's why a protocol turns confusing and you may found lost on which instruction should be helpful.

On the basis on what you describe here, it seems your trying to determine the Kd under equilibrium-binding conditions and that's what I mostly use. Here you go a protocol for direct binding; I hope this helps.

Hi Giovane ;

Thank you so much for your useful protocol. I'm going to do it during next few days and will inform you about the results. I searched for "yeast binding assays"(as you mentioned in step 7) and found some papers. It would be very kind of you if you could explain more if there is any practical point with this assay.

Thanks