Chamberlain Lab

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Historically, cell labelling with [3H]palmitic acid has been the principle method used for the detection of S-acylated proteins. However, this approach lacks sensitivity (exposure times can be on the order of months!) and has obvious safety implications. Latest technical developments in the field have seen the wider use of more sensitive approaches for studying S-acylation including ‘click chemistry’, Acyl-Biotin Exchange (ABE), and Acyl-Resin-Assisted Capture (acyl-RAC).

Recently, we have replaced [3H] palmitate labelling with Click Chemistry as the method of choice for studying S-acylation leading to a significant increase in experimental throughput as a result of increased sensitivity. The click chemistry approach involves labelling cells with azido or alkynyl derivatives of palmitic acid for times ranging from 15 min to 4 hours. These palmitic acid derivatives are faithfully incorporated into S-acylated proteins, which are subsequently conjugated to a reporter molecule (typically a fluorescent tag) via copper-catalysed azide alkyne cycloaddition (CuAAC).

 

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The described click chemistry approach is highly amenable to isolated cells or cell lines, but is less suited for the detection of S-acylated proteins in intact tissues. The most common method for capturing S-acylated proteins from tissue (e.g. brain) was ABE, but this is being replaced by acyl-RAC, which is a simpler and more efficient technique. Acyl-RAC exploits the labile nature of the thioester bond, joining acyl chains to cysteine residues, to specifically capture S-acylated proteins from cell or tissue extracts. Briefly, following cell lysis, free sulfhydryl groups are blocked with MMTS, and the lysate is then treated with the nucleophile hydroxylamine to cleave thioester bonds, with the subsequent capture of proteins containing free cysteine thiolates on thiopropyl sepharose beads

 

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