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5.6K Views
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13:46 min
July 11th, 2020
DOI :
10.3791/61262-v
Chapters
0:00
Introdcution
0:48
Transfection of HEK293 cells
1:49
Media Collection and Storage
3:02
Media Concentration and Purification
4:58
In vitro Mono-biotinylation Reaction
6:01
Biotinylation Analysis
6:56
Visualization of Retrograde Axonal Transport of BDNF-QD in Live Neurons
7:58
Represetative Results
13:17
Conclusion
Transcript
该程序的总体目标是在涉及 HEK293 细胞转染和色谱柱纯化的优化协议中生产和纯化 BDNF 2 管。然后,BDNF oviduct 通过体外反应进行单生物素化,用于后标记和荧光显微镜检测。该程序的主要优点是,它允许BDNF的生产、纯化和单生物基化得到优化。
妊娠参数已针对使用经济高效的转染试剂进行最大 BDNF 生产进行了优化,纯化过程在色谱设置中执行,便于操作,并允许升级过程。首先稀释60微克聚乙胺胺在500微升未升制的DMEM和20微克的BDNF卵巢质粒在500微升未升D
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Summary
含有 Avi 序列 (BDNFAvi) 的重组 BDNF 以经济高效的方式在 HEK293 细胞中生产,并通过亲和色谱进行纯化。然后,BDNFavi 与管中的酶 BirA 直接单生物素化。与市售的BDNF相比,BDNFavi和单生物素化BDNFavi保留了其生物活性。
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