Author: Bederka, Lydia H.; Bonhomme, Cyrille J.; Ling, Emily L.; Buchmeier, Michael J.
Title: Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization Document date: 2014_10_28
ID: wbh06gzb_35
Snippet: Coimmunoprecipitation. Transfected HEK 293T cells were lysed using 0.1% NP-40 lysis buffer (200 mM Tris [pH 6.8], 150 mM NaCl, 1 mM EDTA) with fresh protease inhibitors (RPI Corp.). Clarified lysates were incubated with protein G beads (GE Healthcare) for 30 min at 4°C and collected by mild centrifugation (8, 200 ϫ g, 4°C, 30 s) prior to overnight incubation with either anti-HA agarose beads (Sigma) or the GP1-specific 2.11-15 antibody at 4°C.....
Document: Coimmunoprecipitation. Transfected HEK 293T cells were lysed using 0.1% NP-40 lysis buffer (200 mM Tris [pH 6.8], 150 mM NaCl, 1 mM EDTA) with fresh protease inhibitors (RPI Corp.). Clarified lysates were incubated with protein G beads (GE Healthcare) for 30 min at 4°C and collected by mild centrifugation (8, 200 ϫ g, 4°C, 30 s) prior to overnight incubation with either anti-HA agarose beads (Sigma) or the GP1-specific 2.11-15 antibody at 4°C with continuous end-over-end rotation (39) . With the GP1 immunoprecipitation samples, after overnight incubation protein G agarose beads were added to each sample for 1 h at 4°C. Beads were washed thrice with 0.1% NP-40 wash buffer and once without detergent.
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