Aug 1, 2017 · To evaluate S-fatty acylation of endogenous proteins as well as to determine the number of S-fatty acylation events, we developed the acyl-PEG exchange (APE) that utilizes cysteine-specific chemistry to exchange S-fatty acylation sites with mass-tags of defined size, which can be readily observed by western blotting.

Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

To assess the levels of S-fatty acylation of endogenous proteins, we have developed the acyl-PEG exchange (APE), which selectively replaces S-fatty acids with maleimide-functionalized polyethylene glycol (mPEG-mal) reagents of defined mass.

Apr 4, 2016 · To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE).

Jun 5, 2017 · Acyl PEG Exchange (APE) analysis of HEK293 cells expressing HA-HRas revealed the unmodified protein and two band shifts indicating two palmitoylation sites, in agreement with the putative sites of palmitoylation (Figure 2).

Sep 20, 2024 · Here, we present an optimized acyl-PEGyl exchange gel shift (APEGS) assay to monitor palmitoylation of high-molecular-weight proteins from primary neuronal cultures. We describe steps for culturing cortical neurons from rat embryos and expressing proteins of interest.

Feb 13, 2018 · To evaluate S-fatty acylation of endogenous proteins as well as to determine the number of S-fatty acylation events, we developed the acyl-PEG exchange (APE) that utilizes cysteine-specific chemistry to exchange S-fatty acylation sites with mass-tags of defined size, which can be readily observed by western blotting.

Apr 26, 2021 · This method is known as the acyl-PEG exchange assay (Howie et al., 2014; Percher et al., 2016). In this method, PEGylation gives a mass shift that can be detected by SDS-PAGE and western blot. Acyl-PEG exchange can not only quantify the number of acylation sites, but also show the relative amount of protein in each acylation state.

Aug 1, 2017 · To assess the levels of S-fatty acylation of endogenous proteins, we have developed the acyl-PEG exchange (APE), which selectively replaces S-fatty acids with maleimide-functionalized polyethylene glycol (mPEG-mal) reagents of defined mass.

Jun 6, 2016 · Hang and co-workers have developed a simple and efficient method for the proteomic investigation of protein S-fatty acylation. “Acyl–PEG exchange” (APE) replaces palmitoylthioesters with mass tags, which can be visualised by simple gel electrophoresis.