| Assay Method Information | |
| | Microscale Thermophoresis Assay |
| Description: | MST measurements were performed with a Monolith NT.115Pico device (NanoTemper Technologies). JH2 domain protein (triple mutant W659A, W777A, F794H, or triple mutant W777A, F794H, V617F) was fluorescently labeled with the His-tag labeling kit RED-tris-NTA 2nd generation. All dilutions were prepared with a buffer composed of 20 mM HEPES pH 8.0, 150 mM NaCl, 5% glycerol, and 0.05% Tween. Protein was labeled by incubating a mixture of 150 nM protein and 50 nM dye for 30 min at ambient temperature. MST measurements were performed with protein and dye concentrations adjusted to 30 nM and 10 nM, respectively. The serial dilution of 1 and 10 ranged from 160 μM to 0.00488 for 12 from 20 μM to 0.00488 and for 15 from 20 to 0.00061 μM. The serial dilution of 1 and 10 ranged from 160 μM to 0.00488 and for 12 from 20 μM to 0.00488 μM. All measurement samples contained a constant DMSO concentration of 3%. Measurements were performed with standard capillaries (mutant W659A, W777A, F794H) or premium capillaries (mutant W777A, F794H, V617F), medium MST power, and 5% excitation power at ambient temperature. All measurements were performed in triplicate, and were analyzed with the MO.Affinity Analysis software (NanoTemper). |
| Affinity data for this assay | |
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