I have carried ITC experiment a number of times and no matter how much I tried to improve on the experimental design, I just keep getting this poor isotherm (in the attachment) with few points at the beginning of the titration. Here I am investigating the binding affinity of a 15k protein which is supposed to have two binding sites for another protein of 100kDa. The 15kDa protein was the titrant in the syringe at a concentration of 750microM (injection volume is 2microlit.) while the 100kDa protein was in the cell at a concentration of 40microM (protein solubility is a problem and perhaps cannot be higher than 90microM). I am of the opinion that the 15K protein, although has two binding sites for the other protein, might not be able to accommodate two molecules of the other protein due to steric hinderance problem.
My concerns are:
1. The shape of my isotherm (I am unable to tell the stoichiometry of binding from the isotherm) and the few points I have at the beginning of the titration.
2. Why do I have N values of 0.0165 (this looks ridiculously small and the error value is so large) and 0.658?
3. Can I rely on any of the K values?
How do can I improve this experiment?
According to your information, you have protein A with two binding sites for protein B, and you are injecting protein A into protein B.
The direct standard titration would be injecting B into A. The one you are carrying out is the reverse standard titration. In general, you cannot expect the same binding isotherm in both experiments. The reverse isotherm may show unconventional features and cannot be interpreted as two sequential, independent or cooperative, binding events.
For example, in the reverse titration in the first injections you are injecting protein A into an excess of protein B, and, therefore, there will be saturation of both binding sites in protein A, even though the binding affinities could be quite different. As the titration progresses, there will be more and more protein A added, and dissociation of previously bound protein B to associate newly added protein A may occur, especially if negative cooperativity is exhibited by protein A or the binding sites in A have different binding affinities.
Therefore, you cannot analyze the data with a model for direct titrations and you cannot interpret the apparent inflection points as stoichiometries or equivalence points.
According to your information, you have protein A with two binding sites for protein B, and you are injecting protein A into protein B.
The direct standard titration would be injecting B into A. The one you are carrying out is the reverse standard titration. In general, you cannot expect the same binding isotherm in both experiments. The reverse isotherm may show unconventional features and cannot be interpreted as two sequential, independent or cooperative, binding events.
For example, in the reverse titration in the first injections you are injecting protein A into an excess of protein B, and, therefore, there will be saturation of both binding sites in protein A, even though the binding affinities could be quite different. As the titration progresses, there will be more and more protein A added, and dissociation of previously bound protein B to associate newly added protein A may occur, especially if negative cooperativity is exhibited by protein A or the binding sites in A have different binding affinities.
Therefore, you cannot analyze the data with a model for direct titrations and you cannot interpret the apparent inflection points as stoichiometries or equivalence points.
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