So I am currently titrating a ligand (A, 12 µM) which is a dimer (in syringe) to a monomeric protein in cell (B, 61 µM). The experimental conditions are 20 mM Sodium Phosphate, 100 mM NaCl, and 2 mM TCEP at 20 C. I observe reasonable heat change after titration but the stoichiometry is consistently lower than 1. Though on the literature, I have observed a N~1 for the same binding proteins (though the titrations are done in reverse orientation).
I believe it is due to the low C value that I am not observing a sigmoidal binding curve. Unfortunately, I am limited by protein and can not further increase the concentration without precipitation of my protein. the
Would a low N value mean only a fraction of my protein in the cell are binding competent/active? If so is there any way to diagnose using any other method and correct it? Below are the attached thermograms and binding isotherm for more clarity.
Thank you.
The numbers for the concentrations in your text do not agree with those observed in the figure.
Since the ligand is a dimer, would the dimer bind one single molecule of the other protein, or two?
When dealing with dimeric proteins, I always recommend, if possible, to perform the assay with the dimeric protein in the cell, to avoid additional problems.
If you have information about that interaction in a certain experimental scheme, I would recommend following the same experimental scheme. After you reproduce the known behavior, then you can explore other possibilities.
Regarding the c-value: c-value is not a goal in itself. Forget about pursuing a high enough c-value guaranteeing a sigmoidal isotherm. Very often it is not achievable (low affinity), and very often it is not necessary for an experiment to be considered good (there are many other features people pay little attention that are even more important than the c-value).
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