May 30, 2023
GFP-ATG3 GUV Assay
- lmstrong 1
- 1UC Berkeley
Protocol Citation: lmstrong 2023. GFP-ATG3 GUV Assay . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm2d5ng3p/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 30, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82660
Keywords: ASAPCRN, atg3 guv assay, lc3 lipidation, atg3, guv, assay, gfp, atg3 guv
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000350
Abstract
LC3 lipidation on GUVs
Troubleshooting
GUV Preparation
Clean the coverglass
Coat cleaned coverslips with 100 μL 5% (w/w) polyvinyl alcohol (PVA) with a molecular weight of 145,000 (Millipore). Place the coated coverslip in a heating incubator at 60 °C to dry the PVA film for 30 min.
Spread a lipid mixture at 1 mg/ml with a molar composition of 70% DOPC, 20% DOPE, 5% DO-PI(3)P, 5% DOPS, 0.3% Atto647N DOPE uniformly onto the PVA film.
Put the lipid-coated coverslip under vacuum overnight to evaporate the solvent.
Use 100 μL 330 mOsm sucrose solution for swelling for 1 h at room temperature
Harvest the GUVs and use them with 12 h.
GUV Assay
Set up the reaction in an eight-well observation chamber (Lab Tek) at room temperature.
Coat the chamber with 1 mg/ml β casein for 10 min.
Wash the coated chamber with reaction buffer (25 mM HEPES at pH 7.4, 150 mM NaCl and 1 mM TCEP).
Make a 120 µL reaction mixtures with the proteins and 5 mM ATP and 1 mM MgCl2. The final concentration of WIPI2d 200 nM, E3 complex is 50 nM, ATG7 is 100 nM, GFP-ATG3 or Mutant is 100 nM, and mCherry-LC3B is 500 nM.
Add 3 µL GUVs to initiate the reaction.
Pick random views for imaging within 5 min.