May 03, 2024

Public workspaceExpression and purification of Syp-VAMP2 complex into native nanodiscs

DOI
dx.doi.org/10.17504/protocols.io.4r3l2qo24l1y/v1
  • 1Nanobiology Institute, Yale University, West Haven, CT, USA;
  • 2Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA;
  • 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Caroline Brown
Open access
DOI: dx.doi.org/10.17504/protocols.io.4r3l2qo24l1y/v1
Protocol Citation: Caroline Brown, Snehasish Ghosh, Kallol Gupta 2024. Expression and purification of Syp-VAMP2 complex into native nanodiscs. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2qo24l1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 03, 2024
Last Modified: May 03, 2024
Protocol Integer ID: 99219
Abstract
This is a protocol for the purification of Syp-VAMP2 complex into native nanodiscs.
Mammalian expression constructs
Mammalian expression constructs
2d
Thaw Expi-HEK293 cells and passage 3 times
Using a T2A polycistronic vector containing the VAMP2 and Synaptophysin (Syp) genes with FLAG tag, express VAMP2 and Syp in Expi-HEK293 cells using ExpiFectamine transfection reagent for Duration48:00:00 hours.

2d
Spin down transfected cells and rinse in ice-cold PBS.

Solubilization of VAMP2-Syp into native nanodiscs
Solubilization of VAMP2-Syp into native nanodiscs
4h 25m
Resuspend cells expressing VAMP2-Syp into lysis buffer (Concentration50 millimolar (mM) Tris HCl pH 7.4 , Concentration150 millimolar (mM) NaCl , Concentration10 % volume glycerol ) supplemented with protease inhibitor tablets.

Lyse cells using nitrogen cavitation (600 psi, Duration00:15:00 minutes).

15m
Remove debris and nuclei using a 4000 rpm centrifugation spin for Duration00:10:00 minutes.

10m
Ultracentrifuge the clarified lysate at 200,000xg for Duration01:00:00 hour at Temperature4 °C to collect membranes.

1h
Resuspend membranes in 1.5% polymer solution (ChloroSMA80) and incubate at Temperature4 °C for Duration02:00:00 hours.

2h
Post-solubilization centrifuge the sample at 200,000xg for Duration01:00:00 hour to remove insoluble material.

1h
Native nanodisc enrichment using affinity chromatography
Native nanodisc enrichment using affinity chromatography
Incubate native nanodiscs containing VAMP2-Syp complex with anti-FLAG resin DurationOvernight at Temperature4 °C with rotation.

Remove supernatant and wash beads extensively with Concentration50 millimolar (mM) Tris HCl pH 7.4 , Concentration150 millimolar (mM) NaCl , Concentration10 % volume glycerol .

Elute nanodiscs using buffer from Step 11 containing 3XFLAG peptide at 10 ug/ml concentration.