GUV preparation
Chunmei Chang, Liv Jensen
Published: 2022-05-26 DOI: 10.17504/protocols.io.3byl4b398vo5/v1
Abstract
GUV preparation for membrane tube assay application
Steps
1.
GUV Preparation
1.1.
Clean the coverglass.
1.2.
Coat cleaned coverslips with 100 μL 5% (w/w) polyvinyl alcohol (PVA) with a molecular weight of 145,000 (Millipore).
1.3.
Place the coated coverslip in a heating incubator at 60 °C to dry the PVA film for 30 min.
1.4.
Spread a lipid mixture with a molar composition of 70% DOPC, 20% DOPE, 5% DO-PI(3)P, 5% DOPS, 0.3% Atto647N DOPE, 0.01% PEG2000-biotin-DSPE at 1 mg/ml uniformly onto the PVA film.
1.5.
Put the lipid-coated coverslip under vacuum overnight to evaporate the solvent.
1.6.
Use 100 μL 320 mOsm sucrose solution for swelling for 1 h at room temperature
1.7.
Harvest the GUVs and use them with 12 h.
