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An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons
JoVE Journal
Neuroscience
Author Produced
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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
DOI:

13:46 min

July 11, 2020

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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

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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