| Assay Method Information | |
| | DNA Methylation Assay |
| Description: | Assays (0.1 ml) were conducted in 96-well black half-area plates in either a Wallac VICTOR2 or Biotek Synergy Neo plate reader at 37 °C in 10 mM Tris, pH 7.5, 100 mM potassium glutamate, 1 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA, and 5% glycerol. Assays contained varying amounts of oligonucleotide 8006 (5'-FAM-CCTATGCGmCATCAGTTTTCTGATGmCGmCATAGG-3'-Iowa Black, in which mC denotes 5-methyldeoxycytidylate residues (Integrated DNA Technologies, Coralville, IA), AdoMet (HPLC-purified, Sigma), and small molecule inhibitors (5-azaC, Sigma; SGI-1027, generous gift of Dr. Jian Jin, University of North Carolina; LCA, TCI America). Other anthraquinone compounds examined were purchased from ChemBridge Corp, San Diego, CA (UI1055 is N,N-diethyl-1-nitro-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide; UI1060 is ethyl N-{[1-(butylthio)-9,10-dioxo-9,10-dihydroanthra-cen-2-yl] carbonyl}glycinate; UI1061 is 1-(butylsulfonyl)-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid) and Sigma (anthraquinon and anthraquinone 2-carboxylic acid). All assays were conducted in triplicate and contained either human Dnmt1 (621-1600), human Dnmt1 (351-1600), human Dnmt3a (590-912), or M.SssI methyltransferase (New England Biolabs) and 0.8 units of Gla I (Sibenzyme, West RoxRoxbury, MA), except for the Gla I control assay, which did not contain a methyltransferase. Data were fitted using Prism (GraphPad Software, Inc). |
| Affinity data for this assay | |
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