{"product_id":"neb-m0616s","title":"New England Biolabs, M0616S, FTO RNA Demethylase","description":"\u003cb\u003eRelated Categories\u003c\/b\u003e RNA Library Prep for Illumina,, RNA Modification  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eUnit Definition\u003c\/b\u003e A 25 μl reaction in FTO Reaction Buffer containing 100 ng of a 1700-mer N6A methylated Cluc mRNA and 0.5 ug of FTO RNA Demethylase incubated for 1h at 37 °C results in \u0026gt;50% demethylation of the substrate mRNA as determined by LCMS.  \u003cb\u003eReaction Conditions\u003c\/b\u003e 50 mM Tris HCl, pH 7.5 1 mM αKG 1 mM sodium L-ascorbate 75 mM FE(II) Incubate at 37 °C for 1 hour.  \u003cb\u003eStorage Buffer\u003c\/b\u003e 20 mM Tris-HCl 50 mM NaCl 50% Glycerol 0.1 mM TCEP  \u003cb\u003eHeat Inactivation\u003c\/b\u003e No  \u003cb\u003eMolecular Weight\u003c\/b\u003e Apparent: 59104.73 g\/mol  \u003cb\u003eUnit Assay Conditions\u003c\/b\u003e 1 μg FTO RNA Demethylase is incubated with 100 ng of an Cluc mRNA substrate (1766 mer; 100% m6A), 50 mM Tris HCl, pH 7.5, 1 mM αKG, 1 mM sodium ascorbate, and 75 μM Fe(II) in 25 μL reaction at 37 °C for 1 hour. The reaction is then treated with 0.8 units of Proteinase K, Molecular Biology Grade (P8107) at 37°C for 1 h. The product RNA is purified using Monarch ® RNA Cleanup Kit (T2040) then digested to nucleosides using the Nucleoside Digestion Mix (M0649). The extent of m6A demethylation is determined by monitoring the various adenosine species on an Agilent 1290 Infinity II UHPLC equipped with G7117A Diode Array Detector and 6135 XT MS Detector, on a Waters XSelect HSS T3 XP column (2.1 × 100 mm, 2.5 μm) with the gradient mobile phase consisting of methanol and 10 mM ammonium acetate buffer (pH 4.5). The identity of each peak is confirmed by mass spectrometry. The relative abundance of each nucleoside is determined by the integration of each peak at 260 nm or their respective UV absorption maxima.  \u003cb\u003eFAQ\u003c\/b\u003e Q: Are there any unreacted hm6A and f6A products at the end of the FTO RNA Demethylase reaction? A: Yes, ~ 20% hm6A and ~ 10% f6A are detected at the end of the reaction of FTO RNA Demethylase with 100 ng of a Cluc mRNA substrate (1766 mer; 100% m6A). Q: What is the activity of FTO RNA Demethylase in other NEB buffers? A: Tris HCl, pH 7.5 supplemented with 1 mM αKG, 1 mM L-Asc, and 75 μM Fe(II) is the optimal reaction buffer for FTO RNA Demethylase (86% conversion of m6A). The relative activity of FTO RNA Demethylase with NEB Buffers supplemented with 1 mM αKG, 1 mM L-Asc, and 75 μM Fe(II), results in the following % conversion of m6A. Buffer % Conversion 50 mM Tris HCl, pH 7.5 86 rCutSmartTM Buffer 24 NEBuffer 1.1 73 NEBuffer 2.1 24 NEBuffer 3.1 4 Q: What are Enzymes for Innovation? A: Enzymes for Innovation (EFI) is a project initiated by NEB to provide unique enzymes to the scientific community in the hopes of enabling the discovery of new and innovative applications. These enzymes have interesting properties and unique specificities that are not commercially available elsewhere at the quality that you would expect from NEB. Enzymes for Innovation graduates are enzymes that have transitioned from having unknown or exploratory applications to becoming the cornerstone of a defined application. If you have an idea for an “Enzyme for Innovation” with a suggested application that may be useful, please email us at EnzymesForInnovation@neb.com. For more information, please view video. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835478429865,"sku":"M0616S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m0616s","provider":"Iright","version":"1.0","type":"link"}