Author: te Velthuis, Aartjan J.W.; van den Worm, Sjoerd H. E.; Snijder, Eric J.
Title: The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension Document date: 2011_10_29
ID: tx0lqgff_9
Snippet: Bacterial pellets were thawed on ice, resuspended in buffer A [20 mM HEPES pH 7.4, 10 mM imidazole, 0.05% Tween-20, 5 mM b-mercaptoethanol and EDTA-free protease inhibitor cocktail (Roche)] containing 500 mM NaCl, and lysed by sonication. The supernatant was cleared by ultracentrifugation at 20 000 g for 30 min and subsequently incubated with Talon beads (Clontech) for 2 h at 4 C. The beads were washed four times 15 min with 20 volumes of binding.....
Document: Bacterial pellets were thawed on ice, resuspended in buffer A [20 mM HEPES pH 7.4, 10 mM imidazole, 0.05% Tween-20, 5 mM b-mercaptoethanol and EDTA-free protease inhibitor cocktail (Roche)] containing 500 mM NaCl, and lysed by sonication. The supernatant was cleared by ultracentrifugation at 20 000 g for 30 min and subsequently incubated with Talon beads (Clontech) for 2 h at 4 C. The beads were washed four times 15 min with 20 volumes of binding buffer. Ultimately, the C-terminally His 6 -tagged proteins were eluted with 150 mM imidazole in buffer A containing 150 mM NaCl, or cleaved off of the column during a 3-h digestion with SARS-CoV nsp5 in the presence of 4 mM MgCl 2 .
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