Aug 26, 2023
In vitro depalmitoylation assay
- 1Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
Protocol Citation: wusj, schekman 2023. In vitro depalmitoylation assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x9bklqe/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: August 03, 2023
Last Modified: August 26, 2023
Protocol Integer ID: 85896
Funders Acknowledgement:
Randy Schekman
Grant ID: Howard Hughes Medical Institute
Abstract
This protocol describes the in vitro depalmitoylation assay of DNAJC5
In vitro depalmitoylation assay
In vitro depalmitoylation assay
HEK293T, MDA-MB-231, or Hela cells were transfected with plasmids encoding DNAJC5 by lipofectamine 2000 followed the commercial protocol by Thermo Fisher.
Cellular membranes were prepared as described in "Membrane and cytosol fractionation protocol"
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 Overnight in the presence of a cocktail of protease inhibitors
The mobility of DNAJC5 was examined by SDS-PAGE followed by immunoblot.