Visualising N-azidomyristoylated proteins

Parasites were labelled with 50 μM 12-azidododecanoic acid (azidomyristate) for 6 h in either RTH/FCS or DMEM/FCS. Extracts from 5 × 10⁷ cells were made in trypanosome lysis buffer (50 mM Tris-HCl, pH 7.4,150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100 and a cOmplete mini EDTA-free protease inhibitor cocktail tablet) by incubating on ice for 1 h followed by biological inactivation by freeze thawing three times. N-azidomyristoylated proteins from lysates were labelled with IRDye 800CW alkyne, using the Click-iT^® protein reaction buffer kit (Molecular probes)³¹. Lysates were separated by SDS-PAGE on a 4–12% NUPAGE in MES SDS buffer (Invitrogen), and treated with 1 M NaOH for 1 h to remove S- and O- myristoylation¹⁷. Gels were imaged by in-gel fluorescence with an Odyssey SA infrared imager (LI-COR Biosciences). For the inhibition of protein synthesis, parasites were pre-incubated with 50 μg ml⁻¹ cycloheximide for 30 min prior to labelling with azidomyristate. Fluorescence intensity profiles were extracted using ImageJ (http://imagej.nih.gov/ij/) and plotted as a factor of distance migrated through the gel.