Author: Bouvette, Jonathan; Korkut, Dursun Nizam; Fouillen, Aurélien; Amellah, Soumiya; Nanci, Antonio; Durocher, Yves; Omichinski, James G.; Legault, Pascale
Title: High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension Document date: 2018_12_6
ID: 012ipcdr_42
Snippet: A 3-μL drop of 50 nM human WT Dicer in 20 mM HEPES, pH 7.5, 150 mM KCl, 3 mM EDTA, 1 mM DTT, and 2.5% glycerol was applied onto a carbon-coated copper grid previously glow-discharged using an ELMO glow discharge system (Cordouan Technologies, France). After 1 min, excess liquid was blotted and stained for 1 min with 1.5% freshly prepared uranyl formate (Electron Microscopy Sciences, PA). Samples were imaged using a FEI Tecnai T12 (Eindhoven, The.....
Document: A 3-μL drop of 50 nM human WT Dicer in 20 mM HEPES, pH 7.5, 150 mM KCl, 3 mM EDTA, 1 mM DTT, and 2.5% glycerol was applied onto a carbon-coated copper grid previously glow-discharged using an ELMO glow discharge system (Cordouan Technologies, France). After 1 min, excess liquid was blotted and stained for 1 min with 1.5% freshly prepared uranyl formate (Electron Microscopy Sciences, PA). Samples were imaged using a FEI Tecnai T12 (Eindhoven, The Netherlands) Transmission Electron Microscope (TEM) equipped with a LaB6 filament and operated at an acceleration voltage of 120 kV. Micrographs were collected at defocus between 1 and 3 μm on a FEI Eagle 4 k × 4 k CCD camera at a magnification of~67,000×. From these micrographs, 125,000 particles were extracted and 2D align using xmipp from Scipion [54] . The particles were classified in a minimum of 2 classes and the 2D volumes were measured using ImageJ [55] .
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