I would like to analyse Kd of different metal ions with molecules ( polymers, proteins, sugars...) I am not sure if techniques as displacement ELISA, Alpha assay, calcium imaging, DPI, DLS, NMR...and many other similar that can be used in experimental design for interaction among molecules, are suitable for such a measurement and which technique would give most accurate, undisputable data.
thank you very much for any kind of suggestion.
Hi , I would try isotermal calorimetry titration for that as well as chemical shift (NMR)
Since you are dealing with different types of biomolecules having different intrinsic properties. The best way to measure Kd value will be Isothermal titration calorimetry (ITC) or surface plasmon resonance (spr). ITC will give you the accurate and undisputable data. It also gives thermodynamic parameters involved with binding or association of the molecules with metal atoms.
One method you could try is a fluorescence-based competitive displacement assay. You mix the metal ion with a fluorescent dye that changes its fluorescence properties when it binds the metal (many such dyes are commercially available). The simplest case is where binding to the metal causes the fluorescence to increase. The next step is to add the substance you think might be binding to the metal, at a range of concentrations. In our example, if the metal binds to the test substance, the fluorescence of the dye will decrease. The fluorescence change can be plotted against the concentration of the test substance to measure an apparent Kd.
Another good method, if you have access to the equipment is isothermal titration calorimetry. It will give you a true Kd as well as deltaH and deltaS of binding.
Thank you all,
@adam Method that you describe is similar to this one?
http://www.sciencedirect.com/science/article/pii/S0003267097000421
Hi Stefan, I was thinking more in terms of these reagents:
https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/indicators-for-ca2-mg2-zn2-and-other-metal-ions/fluorescent-indicators-for-zn2-and-other-metal-ions.html
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