AviTag™ 技术— 用于蛋白纯化、固定、检测的生物素化标记

点击了解即用型ORF克隆，提供大肠杆菌、哺乳动物细胞、昆虫细胞、酵母和麦胚无细胞表达克隆载体。

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AviTag™是Avidity的商标，AviTag™技术（美国专利号5,723,584、5,874,239和5,932,433）由科罗拉多州丹佛的Avidity有限公司所有。

Figure2. 转染带有AviTag和eGFP的载体或AviTag- IkBα表达克隆到293T细胞，以未转染的细胞为对照，所有细胞均加入纯化的生物素连接酶、生物素(50M) 、ATP(10mM)。 的提取物的混合纯化的生物素连接酶，生物素（ 50M ）和ATP （ 10mM的）如该图所示。混合物在30℃孵育30分钟 （ 1-7、 9-13泳道 ）或4℃孵育30分钟（8、14泳道）后SDS-PAGE结果如图所示。生物素酰化的蛋白通过和链霉亲和素-HRP、化学发光底物(Pierce)结合，可以使用western blot显影。

Example of precipitation of biotinylated AviTag recombinant proteins with streptavidin beads

Figure4. 293T cells were transfected with different combinations of AviTag expression plasmids and biotin ligase, as indicated in the figure. Lysates were prepared 24 hours after transfection and incubated with streptavidin beads for 8 hours. Suspensions were centrifuged and pelleted beads were washed 3 times. Cell lysates (L), supernatants (S) and pelleted biotinylated protein bound to streptavidin beads (B) were resolved by SDS-PAGE. Biotinylated proteins were visualized on a western blot with streptavidin-HRP conjugate and chemiluminescent substrate (Pierce).