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Isothermal Calorimetry (ITC) instrument

The ITC machine measures affinity, stoichiometry, enthalpy change and standard entropy changes for a molecular interaction in a single experiment. The low sample volume means the systems will work with as little as 10µg of protein, which is an important consideration given some users will have limited amounts of material.

ITC measures binding parameters in solution and therefore avoids problems associated with target immobilisation encountered using SPR. The samples used in experiments do not need to be labelled and there are no constraints on the size of proteins/ligands used in the experiments, therefore the machine has utility not only for biologists looking at protein:protein/ligand interactions but is useful for other disciplines e.g. chemistry where the interaction between two small molecules may wish to be investigated.

ITC is particularly suited to people looking at molecular interactions between purified components.

Nanotemper Microscale Thermophoresis (MST) instrument

The MST machine will also measure affinity and stoichiometry, and uses incredibly low volumes/amounts of sample (<4ul as low as 1nM concentration). A major distinction between thermophoresis and ITC is that this machine can measure binding constants with one of the ligands present in a complex mixture such as a cell lysate.

The caveat which further distinguishes it from the ITC is that one of the molecules has to be fluorescently labelled. This might be with a fluorescent dye or GFP for example. Thus this machine will not be suitable for all applications since the addition of a fluorescent tag may alter the biomolecular interaction in certain instances and in these cases ITC will be the more appropriate technology.

The huge advantage of the thermophoresis machine is the ability to quantitate biomolecular interactions in the context of a complex cell lysate using minuscule amounts of sample. This means interactions can be analysed between large macromolecular assemblies such as the ribosome or spliceosome and individual ligands.

Refeyn TwoMP Mass Photometer with MassFluidix device

The Refeyn TwoMP Mass Photometer provides a unique, single molecule method to differentiate between particles of similar size but different mass based on the principle of Interferometric Scattering microscopy. Upon illumination of a sample during a mass photometry measurement, the light scattered by molecules proximal to the measurement surface interferes with light reflected from the measurement surface. Mass photometry quantifies this interference, and the resulting signal exhibits a direct correlation with molecular mass. 
By looking at single molecules, mass photometry provides a unique method to deconvolute mixed populations in samples, providing unprecedented information on composition and homogeneity. Real-time imaging means that changes in molecular interactions, oligomerization and macromolecular assemblies can be followed as they happen, and as all molecules scatter light, it is highly versatile. 
The MassFluidix system HC integrates a computer controlled microfluidic device with the TwoMP Mass Photometer. It raises the upper concentration limit from the nanomolar to the micromolar range to facilitate studies of very low affinity interactions. 

This equipment was funded by a BBSRC ALERT 2024 Mid-range equipment award (BB/Z515930/1).

Contact the Biosciences research team for more information.