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En forbedret protokoll for å rense og direkte mono-bio-biotinylate rekombinant BDNF i et rør for cellulære trafficking studier i nevroner
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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
Chapitres
- 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
Rekombinant BDNF som inneholder en Avi-sekvens (BDNFAvi) produseres i HEK293-celler på en kostnadseffektiv måte og renses av affinitetkromatografi. BDNFavi blir deretter direkte mono-biotinylert med enzymet BirA i et rør. BDNFavi og mono-biotinylated BDNFavi beholder sin biologiske aktivitet sammenlignet med kommersielt tilgjengelig BDNF.
