Hello,
I am having an ITC issue because I cant increase my ligand concentration due to solubility issues.
I know there is past literature on reverse titrations being acceptable. I am more asking about what concentrations I should try for the reverse titration.
I have used fluorescence polarization as a competition based assay showing a Ki of around 10μM for the ligand titrated into protein. Can this help me in determining concentration of protein/ligand for a reverse titration?
I have tried titrating ligand into protein at the maximal concentration of ligand and there is a slight but significant heat change; therfore, Nanoanalyze has suggested I increase ligand concentration -- that is why I amm trying to do a reverse titration now. Thanks for your help.
There is no reason not to perform reverse (protein into ligand) titrations. Molecular weight is not determinant for locating a reactant in string or cell. On the contrary, reactant availability and solubility should be considered when designing the experiment.
If the stoichiometry of interaction is 1:1, there is no complication when performing the reverse titration, and similar results should be obtained with direct and reverse titrations, unless some unspecific phenomena (e.g., self-association of reactant) occur. In fact, this experimental setup is recommended to rule out such kind of unwanted phenomena.
However, when the stoichiometry is 1:n, with several ligand binding sites in the protein, the analysis must be performed carefully and properly, because the results can be misinterpreted. The direct and reverse titration may show very different profiles if the sites are not identical or they show cooperativity. Bear in mind that, in this case, the direct and reverse titrations represent very different pathways along equilibrium triggered by different chemical perturbations.
If the interaction is 1:1, you should plan the experiment with the same concentration you would select in the direct titration:
[syringe] = 10 x [cell]
The actual values to be used are also dependent on the enthalpy of the interaction.
The expected binding affinity may help you in deciding whether or not increasing additionally the syringe concentration to guarantee enough saturation at the en of the titration (important for low affinity).
I forgot to say that when the stoichiometry is 1:n (a protein with n ligand binding sites), for a direct titration the concentrations should be:
[syringe] = 10 x n x [cell]
and for a reverse titration:
[syringe] = 10/n x [cell]
In other words, you must consider the concentration of binding sites, instead of the concentration of protein.
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