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An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons
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An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons
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
Kapitel
- 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
Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.
