Figure 1.

Steps of the APEGS assay

Cells are lysed with Buffer A, followed by sonication and centrifugation (1). Lysates are incubated with 25 mM TCEP for 1 h to reduce disulfide bonds (2); lysates are then treated with 50 mM NEM overnight to block free cysteine residues (3); methanol-chloroform precipitation (MCP) is performed to remove NEM (3–4); palmitate moieties are then cleaved by addition of 2 M NH₂OH (hydroxylamine) for 1 h (4); a second MCP is performed to remove hydroxylamine (4 and 5); formerly-palmitoylated cysteine residues are then labeled with 7 mM MPEG-MAL-10K for 2 h (5); a third MCP is performed to remove excess MPEG-MAL-10K. Samples are then run on a WB after boiling with sample buffer at 95°C for 3 min (6).