Author: Hao, Wei; Wojdyla, Justyna Aleksandra; Zhao, Rong; Han, Ruiyun; Das, Rajat; Zlatev, Ivan; Manoharan, Muthiah; Wang, Meitian; Cui, Sheng
Title: Crystal structure of Middle East respiratory syndrome coronavirus helicase Document date: 2017_6_26
ID: 0vxhgjss_37
Snippet: ATPase assay was carried out as previously described [44] . Briefly, reaction mixtures (50μl) containing 100mM Tris-HCl (pH 8.0), 4mM MgCl 2 , trace amount of [γ-32 P]ATP (~1nM) and the specified amount of ATP (from 10μM to 320μM) were incubated at 30˚C. The reaction was initiated by the addition of MERS-CoV nsp13 (20nM). At each indicated time point, 2μl of quenching buffer (0.5M EDTA) was added to the mixture to stop the reaction. Finally.....
Document: ATPase assay was carried out as previously described [44] . Briefly, reaction mixtures (50μl) containing 100mM Tris-HCl (pH 8.0), 4mM MgCl 2 , trace amount of [γ-32 P]ATP (~1nM) and the specified amount of ATP (from 10μM to 320μM) were incubated at 30˚C. The reaction was initiated by the addition of MERS-CoV nsp13 (20nM). At each indicated time point, 2μl of quenching buffer (0.5M EDTA) was added to the mixture to stop the reaction. Finally, 1μl the sample was spotted on the thin-layer chromatography cellulose TLC plates (Sigma-Aldrich) and resolved with running buffer containing 0.8M acetate and 0.8M LiCl. The plates were dried and analyzed using storage phosphor screen and Typhoon Trio Variable Mode Imager (GE healthcare). ATP turnover was quantified using ImageQuant TL software (GE Healthcare).
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