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SNARE constructs

JJ Junyi Jiao
FH Frederick M Hughson
YZ Yongli Zhang

Last updated date: Mar 5, 2020 Views: 1076 Forks: 0

original An abbreviated version of this protocol was published in eLife in Dec, 2018
Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association
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Dear Sergio,


Thanks for your interest in our work. Sorry for my late reply, as I just found the message being filtered by my mailbox. To generate the syntaxin-VAMP2 conjugate, we purified individual neuronal SNARE proteins, including SNAP-25B. We then formed the ternary SNARE complex, purified the complex, and crosslinked syntaxin and VAMP2 in the complex in an oxidizing condition. Finally, we pulled single-SNARE complexes to dissociate the SNAP-25 molecules to obtain single syntaxin-VAMP2 conjugates. Our experimental procedures were generally described in our elife paper ( Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association. DOI: 10.7554/eLife.41771). We have been developing protocols to prepare the syntaxin-VAMP2 conjugates and the template complexes in bulk. In collaboration with Fred Hughson's group at Princeton Unversity, we are trying to determine the atomic structure of the template complex identified from our single-molecule experiments. We will be happy to share with you our protocol once it is ready.


Please let me know if you have furhter questions


Best regards,


Yongli


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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Jiao, J, Hughson, F and Zhang, Y(2020). SNARE constructs. Bio-protocol Preprint. bio-protocol.org/prep237.
  2. Jiao, J., He, M., Port, S. A., Baker, R. W., Xu, Y., Qu, H., Xiong, Y., Wang, Y., Jin, H., Eisemann, T. J., Hughson, F. M. and Zhang, Y.(2018). Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association. eLife. DOI: 10.7554/eLife.41771

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