Equipment available: Nanotemper Monolith NT.115 Red Blue excitation
Equipment location: New Biochemistry 00-064
Equipment coordinator: Dr David Staunton
Equipment charge: £25/ two hour time slot
Microscale thermophoresis (MST) can quantify the binding affinities of ligands and discriminate between binding sites on the basis of affinity. This method is suitable for studying binding interactions of very small amounts of protein in solution.
The method detects changes in the hydration shell, charge or size of molecules. It allows a wide range of biomolecular interactions to be measured under close to native conditions (any buffer can be used).
An infrared laser generates a microscopic temperature gradient in a sample within a glass capillary in which fluorescence is used to monitor the motion of molecules along the temperature gradient. The solvation entropy and the hydration shell have been identified as key factors that define the movement of molecules along the temperature gradient. This movement is called thermophoresis which is very sensitive to changes in the hydration shell of the protein being observed.
Since ligand binding changes the hydration shell, thermophoresis changes can be used to determine binding affinities. The small volumes (4ul sample in the capillary) and the wide buffer requirements make it very attractive for ligand binding studies with small amounts of material and membrane proteins. Unlike fluorescence polarisation (FP) the label can be on either ligand or receptor regardless of relative size but FP samples using fluorescein-like labels can be used directly in the MST instrument.
The Nanotemper Monolith NT.115 Red Blue instrument can excite fluorescence with wavelengths 460-480nm and 600-650nm and detect between 510-530nm and 675-690nm. This makes it suitable for GFP and Cy5 among other labels.
The solution inside the capillary is locally heated with an IR laser, which is coupled into the fluorescence microscope using an IR reflecting ‘hot’ mirror. Changes to the fluorescent intensity due to thermophoresis are monitored as a function of time until the heating is switched off and the process repeated for the other samples.
We stock standard capillaries (250 per vial £65) and for samples that stick to the standard capillary walls we also have premium capillaries (200 per vial £240).
We recommend users to check the fluorescence of potential labels in the instrument before setting up titrations to ensure that the signal is strong enough at their working concentrations (KD or below). Once a working concentration of label is determined this is kept constant in all samples and the other component is varied to give a titration. Up to 16 capillaries can be run in a single experiment.
The sample volume in the capillaries is 4ul but we recommend working with 20ul samples for ease of loading the capillaries.
As with any titration the concentration range should straddle the KD of the interaction and if unknown a wide dilution range may be required at first to find the appropriate concentrations.
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