The mixing time is a T1 rho filter to reduce the protein background signal and should have little effect on the STD signal. The filter works well for larger proteins, but may have to be increased for smaller proteins.

We typically use the default settings for the saturation pulse train, see the original Mayer & Meyer article (JACS, 2001, 123, 6108-6117) for more details on this. We also select satmode='ny' and depend only on the DPFGSE sequence for water suppression.

Finally, we have found that the experiment works best in D2O.

STD NMR is a powerful and fast method used to study protein-ligand interactions, focusing on the signals of the ligand. Besides, values including KD, the association and dissociation kinetics (Koff and Kon respectively) can be obtained from STD NMR study as well.

STD-NMR is a simple and fast method to screen ligand and characterize protein binding and it does not require expensive stable isotope or radioisotope labeling. Even to monitor low receptor concentration at low molecule concentration, STD can be used via non-scalar magnetization transfer, while NMR spectroscopy requires a much higher concentration of proteins that are labeled stable isotopes. In an STD-NMR experiment (as shown in the figure), the di?erence spectrum is obtained by subtracting a spectrum where the protein is selectively saturated (on-resonance) with signal intensities ISAT from that without saturation (off-resonance) with intensities I0. And the difference in intensities (ISTD=I0-ISAT) indicates molecule binding. Creative Biostructure MagHelix™ Saturation Transfer Differences (STD) NMR, hope helpful.