In vitro depalmitoylation assay
schekman, wusj
Published: 2023-08-26 DOI: 10.17504/protocols.io.bp2l6x9bklqe/v1
Abstract
This protocol describes the in vitro depalmitoylation assay of DNAJC5
Steps
In vitro depalmitoylation assay
1.
HEK293T, MDA-MB-231, or Hela cells were transfected with plasmids encoding DNAJC5 by lipofectamine 2000 followed the commercial protocol by Thermo Fisher.
2.
Cellular membranes were prepared as described in "Membrane and cytosol fractionation protocol"
3.
For chemical deplamitoylation, the membranes were resuspended and incubated with 0.5 M hydroxylamine (pH 7.2) or 0.5 M Tris (pH 7.2, control) at Room temperature in the presence of a cocktail of protease inhibitors
4.
The mobility of DNAJC5 was examined by SDS-PAGE followed by immunoblot.
