Author: Ishimaru, Daniella; Plant, Ewan P.; Sims, Amy C.; Yount, Boyd L.; Roth, Braden M.; Eldho, Nadukkudy V.; Pérez-Alvarado, Gabriela C.; Armbruster, David W.; Baric, Ralph S.; Dinman, Jonathan D.; Taylor, Deborah R.; Hennig, Mirko
Title: RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus Document date: 2012_12_26
ID: zrbn637z_23
Snippet: At 5, 8 and 12 h pi, SARS-CoV and S3L2-ACUucc pk infected or mock-infected cells were washed and lysed in buffer containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.5% deoxycholine, 1% Nonidet P-40, 0.1% SDS and post-nuclear supernatants added to an equal volume of 5 mM EDTA and 0.9% SDS, resulting in a final SDS concentration of 0.5%. Samples were heat inactivated twice before usage. On 4-20% Criterion gradient gels (Bio-Rad), 10 mg of protein wa.....
Document: At 5, 8 and 12 h pi, SARS-CoV and S3L2-ACUucc pk infected or mock-infected cells were washed and lysed in buffer containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.5% deoxycholine, 1% Nonidet P-40, 0.1% SDS and post-nuclear supernatants added to an equal volume of 5 mM EDTA and 0.9% SDS, resulting in a final SDS concentration of 0.5%. Samples were heat inactivated twice before usage. On 4-20% Criterion gradient gels (Bio-Rad), 10 mg of protein was loaded and transferred to a polyvinylidene difluoride membrane. Blots were probed with polyclonal rabbit antisera directed against nsp1 (diluted 1:500) or nsp16 (diluted 1:200) (28) and developed using enhanced chemiluminescence reagents (Amersham Biosciences).
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