Abstract
Protein prenylation is one of the most common post-translational modifications affecting hundreds of eukaryotic proteins. Rab geranylgeranyl transferase prenylates exclusively the GTPases of Rab family, and inhibition of this enzyme induces apoptosis in cancer cells, making it an attractive anticancer target. To efficiently test for possible inhibitors of this enzyme, a robust high-throughput assay is required. Here, we present protocols for the synthesis of a fluorescent analogue of geranylgeranyl pyrophosphate NBD-FPP. We utilized this fluorescent probe to design a high-throughput fluorometric assay of Rab prenylation. This continuous fluorometric assay offers the advantage of being sensitive, cost-effective and amendable to miniaturization. The protocol includes the synthesis of the fluorescent substrate, setup of the assay, assay procedure and data analysis. The procedure for the Rab geranylgeranyl transferase (RabGGTase) plate assay depends on the number of compounds in the screen but generally can be performed within a day.
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Acknowledgements
K.A. was supported by a Heisenberg Award of the Deutsche Forschungsgemeinschaft. L.B. was supported by a Sofja Kovalevskaja Award of the Alexander von Humboldt Foundation. This work was supported in part by grant DFG AL 484/7-2 to K.A. and grant SFB642 of the Deutsche Forschungsgemeinschaft to K.A., Roger S. Goody and Herbert Waldmann.
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Analytical data for the synthesis of a fluorescent analogue of geranylgeranyl pyprophosphate (DOC 54 kb)
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Wu, YW., Alexandrov, K. & Brunsveld, L. Synthesis of a fluorescent analogue of geranylgeranyl pyrophosphate and its use in a high-throughput fluorometric assay for Rab geranylgeranyltransferase. Nat Protoc 2, 2704–2711 (2007). https://doi.org/10.1038/nprot.2007.401
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DOI: https://doi.org/10.1038/nprot.2007.401
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