May 31, 2023
LC3 Lipidation assay for ATG3 Mutants
- 1UC Berkeley
Protocol Citation: lmstrong 2023. LC3 Lipidation assay for ATG3 Mutants. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjxodlmk/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 31, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82662
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000350
Abstract
LC3 lipidation assay on liposomes
Mix 1 μM ATG3 WT or mutant, 1 μM ATG7, 500 nM WIPI2d, 1 μM ATG12–ATG5-ATG16L1, 5μM LC3B
and 0.5 mg/mL extruded liposomes in reaction buffer (25mM HEPES pH 7.4, 150 mM NaCl, 1 mM TCEP).
Incubate at room temperature for 20 minutes.
Quench with 4X LDS loading buffer. Boil samples for 10 minutes at 60ºC. Load and run SDS PAGE gel
Visualize protein bands with coomassie blue