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_12
Snippet: To study SARS-CoV nsp(7+8) complex formation, different nsp8:nsp7 ratios were mixed in binding buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 5% glycerol, 0.1% Triton X-100 and 1 mM DTT) to give a final reaction volume of 10 ml. The proteins were pre-incubated for 10 min at 20 C, after which cross-linking was initiated through the addition of 0.5 ml of a freshly prepared 2.5% glutaraldehyde solution. The reactions were incubated for a further 5 min at 3.....
Document: To study SARS-CoV nsp(7+8) complex formation, different nsp8:nsp7 ratios were mixed in binding buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 5% glycerol, 0.1% Triton X-100 and 1 mM DTT) to give a final reaction volume of 10 ml. The proteins were pre-incubated for 10 min at 20 C, after which cross-linking was initiated through the addition of 0.5 ml of a freshly prepared 2.5% glutaraldehyde solution. The reactions were incubated for a further 5 min at 30 C and then terminated with 1 ml 1 M Tris pH 8.0. Analysis of complex formation was performed on SDS-PAGE gels, which were stained with Coomassie G-250 dye.
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