Dec 02, 2022
LC3-lipidation-assay
- 1University of California, Berkeley
Protocol Citation: Liv Jensen 2022. LC3-lipidation-assay. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg392ypg25/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: December 02, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 73492
Keywords: ASAPCRN, lc3 lipidation, lipidation, synthetic liposome, using purified protein, lc3, purified protein, assay protocol
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Abstract
Protocol for an in vitro LC3 lipidation assay using purified proteins and synthetic liposomes.
Materials
ATG3, ATG7, WIPI2, ATG12–5-16L1-GFP, LC3B
400nm extruded liposomes (70% DOPC, 20% DOPE, 5% DOPS, 5% PI(3)P)
Reaction buffer:20mM Tris pH8.0, 150mM NaCl, 1mM MgCl2, 1mM TCEP,1mM ATP
Mix 2μM ATG3, 2μM ATG7, 1μM WIPI2, 200nM ATG12–ATG5-ATG16L1-GFP, 5μM LC3B
1:1 with extruded liposomes in reaction buffer.
Incubate at 37 ̊C
At 0, 15, 30, 60, and 120 minutes, remove 12μl of reaction mixture andquench by
adding 4ul of SDS-PAGE loading bufferandheatingat 60 ̊C for 10 minutes.