Author: Liu, Hong Yan; Gao, Xiaohu
Title: A Universal Protein Tag for Delivery of SiRNA-Aptamer Chimeras Document date: 2013_11_7
ID: 0atfsivf_22
Snippet: Cells were suspended in Bug-Buster Mix (Novagen) with 5 ml reagent per gram of wet cell paste. Bug Buster Mix was added with protease inhibitor EDTA-free cocktail (Pierce), 10% glycerol, and 1.0 mM THP (Novagen). The cell suspensions were incubated on a shaker platform for 30 min at room temperature. Insoluble cell debris was removed by centrifugation (Beckman TL120) at 20,0003 g for 20 min at 4uC. The soluble extracts were loaded onto affinity c.....
Document: Cells were suspended in Bug-Buster Mix (Novagen) with 5 ml reagent per gram of wet cell paste. Bug Buster Mix was added with protease inhibitor EDTA-free cocktail (Pierce), 10% glycerol, and 1.0 mM THP (Novagen). The cell suspensions were incubated on a shaker platform for 30 min at room temperature. Insoluble cell debris was removed by centrifugation (Beckman TL120) at 20,0003 g for 20 min at 4uC. The soluble extracts were loaded onto affinity columns with Ni-charged His Bind Resin (Novagen). Following washing with binding buffer and washing buffer, the desired proteins were eluted with 6 volume elution buffer (Novagen). The eluted proteins were dialyzed with PBS containing 10% glycerol and 0.1% (v/v) b-mercaptoethanol for 24 hours.
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