| Assay Method Information | |
| | Fluorescence Polarization Assay |
| Description: | The DNA-binding competition assay was performed in 25 μL, in black 384-well plates, using either 30 mM HEPES (N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic Acid) (pH 7.5, with 100 mM KCl, 40 mM NaCl, 10 mM NH4OAc, 10 mM Guanidinium, 2 mM MgCl2, 0.5 mM EDTA, 0.01% NP-40) for mouse full length SOX18, or Tris-NaCl (10 mM Tris pH 8.0 and 100 mM NaCl) for SOX-HMG fragments. All experiments were performed using a 40bp double-stranded Prox1-DNA element with 5' fluorescein amidite (FAM) label (Sigma Proligo or InVitrogen). Optimum binding levels were obtained at 200 nM of mouse full length SOX18 and 60 nM of SOX-HMG fragment, in presence of 5 nM labelled DNA. Controls consisted of free labelled DNA (low FP milli-Polarization index, mP), labelled DNA in presence of protein (negative control, high mP), and labelled DNA and protein in presence of 400 time competing excess of unlabelled DNA (positive control, low mP). Depending on compound, final DMSO concentrations ranged from 2 to 3.33% v/v. After mixing protein, DNA probe and compound, plates were sealed and briskly agitated in the dark for 5 minutes at room temperature, 10 minutes at 37 °C, and 30 minutes at room temperature, before reading fluorescencepolarization on a Tecan M1000Pro (λexc = 485 nm, λem = 525 nm). All experiments were performed intriplicates. |
| Affinity data for this assay | |
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