I am studying the binding of a new ligand with a protein. Can anyone guide me to select the modeling (independent, multiple site, sequential three site, sequential two site) to analyze the ITC data? I also dont know the n value of the ligand. Does n value signify that the ligand has good affinity for the protein? Is it necessary to get n=1 for proper ligand-protein binding?
I also dont know the n value of the ligand---- Assume it 1.
Does n value signify that the ligand has good affinity for the protein?----NO, for affinity you need to calculate Kd.
Is it necessary to get n=1 for proper ligand-protein binding?----NO, generally, try to fit your isotherm on One-site analysis, and if it gave you value close to 1 (0.8 - 1.2), than you can trust the kd and other parameters.
Dear Moumita
difficult to answer without looking at your ITC data.
Usually, for the choice of model, the simpler the better.
Of course, additional data such as SPR, NMR, FP would be a plus in order to confirm your interaction.
I'm not sure what you have in your mind with the n coefficient. Usually the n corresponds to the number of binding sites for an interaction between two molecules. Such a n coefficient doesn't mean high or low affinity. It is merely telling you about the stoechiometry. A value of 1 is the target value for n, assuming you're facing a 1:1 interaction.
Best
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