Upon binding to a ligand, the fluorophore bound to a target molecule undergoes a spectral shift relative to their known peak emission wavelength (solid line) leading to a decrease in the fluorescence at 650 nm (dotted line) and an increase in the fluorescence at 670 nm (dashed line) or vice versa.
The notion of spectral shift isn’t new, but Dianthus and Monolith are the only instruments that use it to derive affinity constants. Shifts in the emission spectrum are observed and measured in solution from a mixture of target and ligand in microwells or glass capillaries.
In a Spectral Shift assay, the molecule you label with the fluorophore is called a target. The other binding partner — another protein, nucleic acid sequence, small molecule, or fragment — is called a ligand.
To calculate the Kd, you mix together a constant amount of the fluorescently labeled target with a dilution series of a ligand. The ratio of the fluorescence intensities recorded in an isothermal environment at 670 and 650nm is plotted against the logarithmic ligand concentration. The Kd is determined from the binding curve using the law of mass action.
NanoTemper instruments resolve even sub-nanometer spectral shifts of the emission wavelength maximum — too low to be measured with a conventional plate reader or fluorimeter — so you can derive binding affinities with high precision.
Get Dianthus if you want a plate-based screening platform that tackles your challenging affinity screenings. Learn more about the instrument and how it works.
Get Monolith if you want to handle nearly all types of molecular interactions — even the most challenging ones — with ultra-low sample consumption. Learn more about the instrument and how it works.