Methodologies to Aid in the Automation of Cell Based Assays: Detection of Protein Palmitoylation on the IN Cell Analyzer 3000
3 July 2013

The covalent attachment of long chain saturated fatty acids occurs on a wide variety of proteins and can dramatically influence protein localisation and function. The two most common modifications involve acylation with 14-carbon (myristate) or 16-carbon (palmitate) fatty acids. Myristylation of proteins typically occurs at the N-terminus in a cotranslational reaction catalysed by Nmyristyltransferase. In contrast, palmitate is attached post translationally to cysteine residues via a thioester bond. Members of the Src and Ras families of oncoproteins function at the inner leaflet of cellular plasma membranes to control biological signal transduction pathways critical for cancer proliferation. These proteins are anchored to plasma membranes by covalently linked lipids, which are attached post-translationally by enzymes including protein myristoyltransferase, protein farnesyltransferase (FTase), and the membrane associated palmitoyl acyltransferase (PAT). To enable the identification of compounds that may inhibit the palmitoylation of SRC proteins Creaser and Peterson have developed a method for the rapid synthesis of a pro-fluorescent cell permeable lipopeptide, that mimics the Myr-Gly-Cys (PAT lipopeptide) Nterminus of Src family proteins. This poster describes a cellular screening assay developed using this probe with the IN Cell Analyzer 3000.

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