| Assay Method Information | |
| | In Vitro Inhibition Assay Method |
| Description: | The in vitro inhibition assay method was adapted from Svenson and Jaffrey, 2016. All reactions were performed in a 96-well plate with 200 μL assay buffer (50 mM HEPES pH 6, 300 μM 2-oxoglutarate, 300 μM (NH4)2Fe(SO4)2 6H2O, 2 mM ascorbic acid in RNase-free water) with 7.5 μm6A7-Broccoli RNA and 0.250 μM FTO. Inhibitors were added in concentrations ranging from 0.008-40 μM; all inhibitors were dissolved in DMSO and added to a final concentration of 0.2% DMSO. Prior to incubation, 40 μL read buffer (250 mM HEPES pH 9.0, 1 M KCl, 40 mM MgCl2, 2.2 μM DFHBI-1T in RNase-free water) was added to bring the final well volume to 200 μL. After incubation at room temperature for 2 hours, the plates were left at 4 C. overnight (16 hours) to allow DFHBI-1T to bind to A7-Broccoli RNA. Specificity assays were performed by the same method with 0.250 μM ALKBH5. Fluorescence intensity was measured with a BioTek Synergy plate reader with FITC filters (excitation 485 nm, emission 510 nm). Sigmoidal dose-response curves were fitted in GraphPad Prism 6. |
| Affinity data for this assay | |
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