Hello,
I am doing ITC and getting repeatable curves that look very promising. After reading some articles, it seems the biggest challenge to avoid is buffer mismatch. Is there a way to rule out that my curve is indeed ligand to macromolecule binding rather than buffer mismatch (i.e. shape/values of curve, shape/values of trace, thermodynamic values)?
I am very careful when preparing my samples but because my ligand is a small molecule I can't dialyze. I am pretty certain my buffers are well matched but I just want to be certain.
Any help would be appreciated.
make samples that are the same except have a different small molecule of nearly the same MW that you know does NOT bind.
that is your reference - if it is buffer mismatch you should get about the same result. If it is binding the result should be much different.
Buffer mismatch results in large, similar heat effects, but not in thermogram profiles similar to this one you are showing.
Although buffer mismatch should be avoided, there are ways to handle it and reduce its deleterious effect on data fitting. Unless the mismatch is too large, usually good estimates of the interaction parameters can be obtained.
Adrian Velazquez-Campoy I am currently using an analog of the ligand that has no catalytic activity as a negative control and it is surprisingly showing similar binding to the active ligand above. This is why I was concerned about buffer mismatch; howerver, when I have preformed ligand into buffer and buffer into protein there is very minimal heat transfer even though everything is prepared exactly the same. Would a reasonable explanation be that because the ligand is organic it might be associating with the protein just due to the hydrophobic effect of disordering water that is caged around the ligand?
The analog with no catalytic activity may bind perfectly well. Remember that catalytic activity requires binding, but binding does not necessarily results in catalytic activity (e.g. an inhibitor).
The fact that the ligand is organic and hydrophobic, and the hydrophobic effect may play a role has nothing to do with your observation. The same could happen with hydrophilic ligands. It is just a matter of one ligand lacking a particular bond or group which is critical for catalysis.
It is much simpler: two similar ligands can bind to a protein, and only one having catalytic activity.
For me it looks fine. In my case I worked with a difficult protein, and I needed to match the 1% DMSO in protein and ligand manually. It is clear when you have a bad profile that comes from the buffer mismatch. Recomendations: always use the same batch of buffer that you use por purification as well. If you need to add a solvent manually, use the same pippete to add the solvent. Don't use old samples for a new run. Sometimes the protein or ligand gets agregated. That's what comes to my mind. I titrated several mutants and ligands, you can handle this very well after some experience. Preprint Molecular Driving Force of a Small Molecule-Induced Protein ...
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