receptor/ligand binding kinetics

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Karmikae
Karmikae's picture
receptor/ligand binding kinetics

Hi to everyone from the newbie !
I'm on a new project and I've never worked with kinetics. I wanted to know your opinion about something. Globally, I'm working with the thyroid hormone receptor and I want to study the effect of a molecule which binds to the same site as the hormone (more or less an analog of the hormone). First, I want to find what is the quantity of receptor needed to acheive the minimal nonsaturable binding of the receptor. How could I do that ? I just want to make sure the idea I had in mind was fine.
Thank you !

Fraser Moss
Fraser Moss's picture
It sounds like you need to

It sounds like you need to perform some 125I-T3 binding studies.

You will need to express you receptor in a cell line of your choosing by transfecting increasing quantities of the plasmid cDNA (starting at 0ng) into the cells and perform binding assays on each transfection condition with a range of T3 concentrations from 0 to a concentrations you expect to be saturating.

There are plenty of protocols for binding assays in the literature which you could tailor to your needs. Once you have established the nonsaturated transfection AND ligand range you can perform competitive binding experiments with your other ligands using these parameters.