I am trying to determine the binding between a protein and cofactor, however the KD value cannot be found online/published. Would the concentrations of my receptor I choose for my assay be more of a trial and error until saturation is reached?
Thank you!
draw a hypothetical analysis according to KD and then use 4 or 5 diffrent concentration ranges closest to the suppositional value of KD
Dear Isabella,
I would suggest you titrate in your protein (receptor) while keeping the fluorescent tracer at a constant concentration. The minimum concentration of the fluorescent tracer will depend on the fluorophore and the sensitivity of your plate reader. Titrating in your receptor will allow you to see how your polarization increases with increasing receptor concentration. Likewise, you can titrate your fluorescent tracer (while keeping your receptor concentration constant), to see which tracer concentration is the most appropriate. Once you have this data, you can choose concentrations of receptor and tracer that will allow you to maximize your assay window (if this is relevant to your experiment).
Best of luck with your experiment,
Roland.
Hi Roland,
Thank you very much. For the concentrations, I choose ones I see fit?
I would suggest to titrate the receptor in a range of 1 nM to 1 µM, which should give you an initial idea about the concentration appropriate for your assay. For the fluorescent tracer, I would suggest 0.1 nM to 200 nM as a first range to try. I hope this is helpful.
All the best,
Roland.
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