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Melanie	时间: 10-23 04:43   主题：无标题
来自：23.236.*.*  QQ：	HA-signal peptide-FLAG-3C-GLP-1R-3C-8×HIS13 human DNGαs46 His6-tagged human Gβ1and Gγ2 were expressed in Tni insect cells （expression systems） using baculovirus as previously described. Cell cultures were grown in ESF 921 serum-free media （expression Systems） to a density of 4 million cells/ml and then infected with three separate baculoviruses at a ratio of 2:2:1 for GLP-1R DNGαs and Gβ1γ2. The culture was harvested by centrifugation 60 h post-infection and the cell pellet was stored at −80 °C. Cell pellet was thawed in 20 mM HEPES pH 7.4 50 mM NaCl 5 mM CaCls 2 mM MgCl2 supplemented with cOmplete Protease Inhibitor Cocktail tablets （Roche） and benzonase （Merk Millipore）. Complex formation was initiated by the addition of 10 μM exendin-4 or 50 μM oxyntomodulin （China Peptides） Nb35-His （10 μg/mL） and apyrase （25 mU/mL NEB）; the suspension was incubated for 1 h at room temperature. The complex was solubilized from the membrane by 0.5% （w/v） lauryl maltose neopentyl glycol （LMNG Anatr
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