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Nanyang Technological University

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

5.7K Views

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13:46 min

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July 11th, 2020

DOI :

10.3791/61262-v

July 11th, 2020

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Nicolás Stuardo¹^,², Guillermo Moya-Alvarado¹^,³, Carolina Ramírez¹^,³, Giampietro Schiavo⁴, Francisca C. Bronfman¹^,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

Summary

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.

Explore More Videos

Recombinant BDNF

Protein Purification

Fluorescence Microscopy

Transfection Protocol

Cellular Trafficking

Incubation Buffer

Protease Inhibitors

Polyethylenimine

Media Components

Chapters in this video

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

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