Author: Lee, Na-Ra; Kwon, Hyun-Mi; Park, Kkothanahreum; Oh, Sangtaek; Jeong, Yong-Joo; Kim, Dong-Eun
Title: Cooperative translocation enhances the unwinding of duplex DNA by SARS coronavirus helicase nsP13 Document date: 2010_7_29
ID: 1k99yv4i_11
Snippet: The chemical cross-linking reagent dimethylsuberimidate (DMS) was used to study the oligomerization of nsP13. A 300 mg/ml DMS stock solution was prepared immediately before use in ice-cold triethanolamine (TEA) buffer (TEA/ HCl, 0.15 M, pH 8.2) and the pH was readjusted to 8.2 with 1 N NaOH. The nsP13 (4 mM) was preincubated in 50 mM TEA buffer (pH 8.2) containing 50 mM NaCl and 5 mM EDTA for 10 min at 22 C and cross-linking was initiated by addi.....
Document: The chemical cross-linking reagent dimethylsuberimidate (DMS) was used to study the oligomerization of nsP13. A 300 mg/ml DMS stock solution was prepared immediately before use in ice-cold triethanolamine (TEA) buffer (TEA/ HCl, 0.15 M, pH 8.2) and the pH was readjusted to 8.2 with 1 N NaOH. The nsP13 (4 mM) was preincubated in 50 mM TEA buffer (pH 8.2) containing 50 mM NaCl and 5 mM EDTA for 10 min at 22 C and cross-linking was initiated by adding DMS (10 mg/ml). After incubation for 1 min at 22 C, the cross-linking reactions were quenched by adding an equal volume of 1 M glycine. The quenched samples were analyzed by SDS-PAGE (4-12% gradient) and proteins were detected by Coomassie Brilliant Blue R-250 staining.
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