Recent studies used microinjection of semisynthetic N-Ras as a means to study real-time spatiotemporal dynamics of palmitoylation and membrane targeting (Rocks et al., 2005 ; microinjection of fluorescent protein allows the analysis of a synchronized (i.e., chemically identical and with the same initial localization) pool of protein. This elegant approach involves coupling chemically synthesized farnesylated peptides encompassing the C-terminal membrane targeting domain of N-Ras to a Cy3-labeled protein consisting of the remainder of N-Ras (amino acids 1-181) via a maleimidocaproyl linker. By using these semisynthetic farnesylated proteins with intact or mutated palmitoylation sites, high-resolution temporal dynamics together with spatial information on cellular palmitoylation and depalmitoylation reactions were open to investigation.