I am happy to report that the key controls worked on the thermofluorimetric analysis protocol I am adapting from the Easley lab’s Analytical Methods paper. The protein does not give any significant signal in the absence of DNA. That’s key: we need to know where the fluorescence is coming from. Additionally, substituting a non-aptamer DNA of the same size does not give significant ΔdF/dT signal at the key temperatures we were tracking for the aptamer-protein complex. So that signal is specific not only to DNA but to aptamer DNA.
The aptamer I’m working with is frustratingly promiscuous. That’s bad. But the thermofluorimetric analysis is consistent with the binding screen with fluorescein-modified DNA.That’s good.
All-in-all, I’ve had more success in 4 days with thermometric analysis than I had in 6 months with fluorescence polarization.