MST Assay Details
In a glass capillary, fluorescent molecules are allowed to interact with ligands in various concentrations. Upon laser beam treatment of a distinct area of the capillary, the intensity of fluorescence changes due to the movement of molecules away from the heated area (thermophoresis, also called thermo-migration), which differs when the ligand is bound. Temperature-induced changes in fluorophore properties may also be observed.
Comparison of a bromodomain inhibitor binding to bromodomain 1 vs bromodomain 2 or BRD4.
MST measurements were performed for two bromodomains of BRD4 protein (BRD4-1 and BRD4-2) in the presence of variable concentrations of the ligand. Determined KDs show a higher affinity of the inhibitor towards the second bromodomain of BRD4.
KD BD1: 2.2E-06M
KD BD2: 8.3E-08M
Instruments: Monolith NT.115 pico (NanoTemper) for the detection of binding affinities in the low pM range with the highest sensitivity.
Turnaround time: The study is typically completed within 2-3 weeks after receipt of materials. Projects are performed on a first-come-first-serve basis.
Sample requirements: Protein purity of 85% or more is required. The minimum protein amount is dependent on the storage buffer and labeling technique. The rough estimate is between 100-200 ug.
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Screening location: The assay is performed in Malvern, PA, USA.