I am trying to measure binding of calcium to an expressed protein we are studying and am getting very large heats of dilution for the calcium solution in my control tests. We prepared a 1 M CaCl2 solution using the solution from our last dialysis step and then that was diluted to 100 mM for the titration using the dialysis buffer. The attached graph shows the raw data and an unfit curve of the 100 mM CaCl2 solution titrated into the dialysis buffer, with no protein present. There is obviously a slight difference in the buffer composition due to the added calcium but this seems like a very large heat of mixing considering that we used buffer from the dialysis for all dilutions. Any ideas what we could do differently to overcome this?
I do not know if you plan to do a protein-calcium titration with a 100 mM solution in the syringe, but then you will need a solution of about 10 mM protein in the cell, which is quite strange, impractical and costly.
The control experiment for the ligand must be done with the same solution you will use in the proper titration with the protein. There is no reason to do a control experiment with 100 mM calcium, and it is not strange at all you get large high injection heat effects. That slight difference between in the composition you mention is the calcium (100 mM), although some other differences could exist.
If you do not know the affinity for the protein-calcium interaction, and the expected stoichiometry is 1:1, a good starting point is to use a 20 μM protein solution for the cell, and a 200 μM calcium solution for the syringe. If the stoichiometry is not 1:1, but 1:n, you should use a 200xn μM calcium solution for the syringe. And if the affinity is low, you should further increase the concentration of the calcium solution.
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