LC3-lipidation-assay
Liv Jensen
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Abstract
Protocol for an in vitro LC3 lipidation assay using purified proteins and synthetic liposomes.
Steps
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.
