| Assay Method Information | |
| | Fluorescence Polarization (FP) Assay |
| Description: | In a flat black bottom 96 well plate (Corning), the buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 20% Glycerol, 0.5 mM TCEP, 0.01% Tween 20) is added—200 μL to column 1 (blank), 295 μL to column 2, 150 μL to columns 3-12. 5 μL of protein (179.0 μM JAK2-JH2-WT, 154.7 μM JAK2-JH2-V617F, 126.3 μM JAK2-JH1) were added to column 2. 150 μL was transferred, using a multichannel pipette, from column 2 to 3, 3 to 4, 4 to 5, until reaching the last column to make a serial dilutions (1:2). 50 μ1 of 24.0 nM tracer were added from columns 2-12 and fluorescence polarization was measured at λexc=485±20 nm, λem=535±25 nm using an Infinite F500 plate reader until no FP variation was observed. From the lowest and highest FP values (tracer free and tracer fully bound to JAK) fraction of ligand bound to the protein to ligand total (Lb/Lt) was calculated for each concentration of the JAK2-JH2-WT, JAK2-JH2-V617F, and JAK2-JH1 (FIGS. 8A-8B). Experiments were carried out by quadruplicates in three independent experiments. The data provided a typical saturation-binding curve and Kd was calculated fitting the results to the Hill equation using Prism 7. |
| Affinity data for this assay | |
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