FROM Greehey CCRI PILOT award
Recently, the Greehey Children’s Cancer Research Institute acquired the Nanotemper MST Monolith high throughput system which is now located in the RNAi/CRISPR Facility. The Monolith now affords researchers a format for high throughput screening to identify small molecules binding to therapeutically relevant proteins, the characterization of protein-drug interactions as well as protein-protein and protein-ligand interactions. The Nanotemper uses microscale thermophoresis to measure affinities of interaction between biologically relevant molecules. Microscale thermophoresis is based on the detection of a temperature-induced change in fluorescence of a target as a function of the concentration of a non-fluorescent ligand. The observed change in fluorescence is based on two distinct effects. In a microscopic thermal gradient, the changes in the migration of a protein when it is bound by a ligand can be detected. In addition, the microenvironment of a fluorophore attached to a protein also becomes sensitive to changes induced by small-molecule interaction. These changes in fluorescent upon ligand binding can be used for determining binding affinities. An additional feature of the MST technology is that the measurements can be performed in solution and does not require the immobilization of targets or ligands to a solid support. An added attraction is that the MST technology is highly sensitive, requiring small quantities of target and ligand and the assays can be measured rapidly.
Capabilities of the Nanotemper Monolith:
- Measurements of dissociation constants, affinity, stoichiometry, and binding energetics are rapid
- MST is equally applicable to a very large size range of molecules. The Monolith instrument can detect interactions between molecules as small as ions to large complexes (e.g. liposomes and ribosomes).
- MST supports measurement under a broad range of solution conditions where measurements are possible in virtually any buffer, including complex detergent mixtures needed for membrane proteins.
- Measurements can be performed under close to native, physiological assay conditions since measurements are possible in serum or cell lysates.
- The method provides reliable diagnostics that make straightforward detection of aggregation and other sample related artifacts possible.
- The method’s unique capillary system ensures low sample consumption, requiring a < 4 μl sample at nM concentration per data point.
- Due to fluorescence technology, MST is extremely sensitive and can reliably measure sub-nM binding coefficients.
- The LED combination (Blue/Red) is suitable for labels most commonly used for labeling macromolecules and suitable for labels commonly used in impure cell extracts.
- Measures dynamics of the interaction of samples labeled in the blue and red fluorescence range.
