Hi,
The ITC protein with ligand showed no saturation pattern in the ITC experiment even after changing both concentrations. The pattern remains the same in the repeated experiments. Is it binding/weak binding/no binding.
There are some issues with your experiment.
1. The concentration in the cell is 1.6 nM, while the concentration in the syringe is 1.7 μM (1000-fold higher!). For a single binding site interaction the concentration in the syringe must be ca. 10-fold higher than that of the cell (I guess you are using a MicroCal instrument). In your case you would be saturating the titrand in the first injections, and whatever you observe is not heat from the interaction, but something unspecific (e.g., ligand dilution). In addition, because you have 1 nM titrand in the cell, assuming a 200 μL cell volume, even if the binding enthalpy were 50 kcal/mol (quite high indeed), the total heat measured in the titration would be 0.01 μcal, and that heat would be distributed over 20 injections. There is no way you will detect that heat (and the interaction) with that concentration.
2. The large heats measured (about 1500-2000 kcal/mol after ligand-concentration normalization) point to a systematic error associated with buffer mismatch between cell and syringe solutions or an effect associated with ligand dilution.
I would suggest to perform a ligand into buffer titration to see the heat of dilution.
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