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Journal / Neuroscience / An Improved Protocol to Purify and Directly…
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Neuroscience
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JoVE Journal Neuroscience
An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons
 

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Article DOI: 10.3791/61262-v • 13:46 min • July 11th, 2020
July 11th, 2020 •
1,2, 1,3, 1,3, 4, 1,3
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

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Summary
Automatic Translation

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.

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Keywords: BDNF Recombinant Purification Mono-biotinylation Cellular Trafficking HEK293 Cells Transfection Chromatography Fluorescence Microscopy Optimization Polyethylenimine Sodium Chloride Imidazole Protease Inhibitors BSA Centrifugation
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