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| Solution Information | help | |
| Enzyme: | DNA (cytosine-5)-methyltransferase 3A | |
| inhibitor: | BDBM46236 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay materials: 1) PTP-PEST (catalytic domain 2-324aa) was produced at the Sanford-Burnham Medical Research Institute's protein production core facility 2) 14LckpCAP394 (pCAP) was purchased from Univ. of Utah peptide synthesis core 3) Assay Buffer = 50mM Bis Tris pH 6.0, 1mM DTT, 0.1% BSA and 0.005% Tween 20. The buffer was filtered prior to addition of enzyme or substrate. 4) Assay was performed in 1536-well flat bottom black plate (Corning, Cat # 3724) Dose response protocol: 1) Using Thermo Scientific MultiDrop Combi, dispense 4 uL of 20 nM PEST working solution to wells in columns 5-48, and Assay Buffer to columns 1-4. 2) Using Labcyte Echo550, dispense 80 nL of serially diluted compounds to columns 5-44 and DMSO to columns 1-4 and 45-48. 3) Using Thermo Scientific MultiDrop Combi, Dispense 4 ul of 72 uM pCAP into all columns 5) Incubate lidded plate for 20 min. at room temp. 6) Read plate on BMG Pherastar reader in fluorescence mode (Ex:340 nm, Em: 460 nm | |
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