| Assay Method Information | |
| | In vitro Enzymatic Activity Assays |
| Description: | In vitro enzymatic activity assays were performed as previously described. The15-mer ssRNA (5'-CUUGUCA(m6A)CAGCAGA-3') was used as a substrate to evaluate the demethylase activity of FTO. All the reactions were carried out in 50 uL of buffer containing 50 mM Tris-HCl pH 7.5, 2.0 uM ssRNA, 2.0 nM FTO, 1.0 mM +/--KG, 280 uM (NH4)2Fe(SO4)2 and 2 mM L-ascorbic acid. CHTB with varying concentrations (1.00 uM, 4.00 uM, 8.00 uM, 16.00 uM, 32.00 uM, 40.00 uM, 64.00 uM, 84.00 uM,164.00 uM and 328.00 u M) were individually added to each reaction mixture and incubated at room temperature for 0.5 h. The reactions were quenched by heating for 5 min at 95 C. The ssRNA was digested by nuclease P1 (1 Unit) and NH4OAc(100 uM, 5 uL) at 42 C for 4 h, followed by the addition of NH4HCO3 (1.0 M, 5 uL), and alkaline phosphatase (0.5 Unit). |
| Affinity data for this assay | |
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