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Un protocolo mejorado para purificar y directamente monobiocalinato recombinante BDNF en un tubo para estudios de tráfico celular en neuronas
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1Department of Physiology, Faculty of Biological Sciences,Pontificia Universidad Católica de Chile, 2Department of Molecular and Cellular Biology, Faculty of Biological Sciences, Center for Aging and Regeneration (CARE UC),Pontificia Universidad Católica de Chile, 3Institute of Biomedical Sciences. Faculty of Medicine and Faculty of Life Sciences,Universidad Andrés Bello, 4Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology and UK Dementia Research Institute at UCL,University College London Campus
Chapters
- 00:00Introdcution
- 00:48Transfection of HEK293 cells
- 01:49Media Collection and Storage
- 03:02Media Concentration and Purification
- 04:58In vitro Mono-biotinylation Reaction
- 06:01Biotinylation Analysis
- 06:56Visualization of Retrograde Axonal Transport of BDNF-QD in Live Neurons
- 07:58Represetative Results
- 13:17Conclusion
BDNF recombinante que contiene una secuencia Avi (BDNFAvi) se produce en células HEK293 de una manera rentable y se purifica mediante cromatografía de afinidad. BDNFavi es entonces directamente monobiontilado con la enzima BirA en un tubo. BDNFavi y BDNFavi monobionicionados conservan su actividad biológica en comparación con BDNF disponible comercialmente.
