Fig. 2.

Detection of ω-alkynyl-palmitoylated proteins labeled with azido-biotin or 3-azido-7-hydroxycoumarin using Cu⁺-catalyzed alkyne-azide cycloaddition. A: A total of 500 ng of purified HMGCS or GAPDH was labeled using 50 μM ω-alkynyl-palmitoyl-CoA or CoA as a negative control as indicated followed by Cu⁺-catalyzed cycloaddition with either azido-biotin (A and B) or 3-azido-7-hydroxycoumarin (azido-coumarin) (C and D). Reactions were separated by SDS-PAGE and reaction products detected on membranes by Western blotting with NA-HRP (A and B) or by direct fluorescence scanning of the polyacrylamide gel (C and D). Corresponding Coomassie blue staining of the membranes or gels is shown on the right (protein staining). Where indicated, enzymes were preincubated in the presence of 10 mM NEM for 30 min prior to additional incubations and Cu⁺-catalyzed cycloadditions.