Abstract
With the proteasome emerging as a therapeutic target for cancer treatment, accurate tools for monitoring proteasome (inhibitor) activity are in demand. In this chapter, we describe the synthesis and use of a fluorescent proteasome activity probe that allows for accurate profiling of proteasomal activity in cell lysates, intact cells, and murine and human patient-derived material, with high sensitivity using SDS-PAGE. The probe allows for direct scanning of the gel for fluorescent emission of the distinct proteasomal subunits and circumvents the use of Western blot analysis. Due to its suitable biochemical and biophysical properties, the fluorescent probe can also be used for confocal laser scanning microscopy and flow cytometry-based experiments.
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Acknowledgements
Henk Hilkmann is acknowledged for peptide synthesis and the authors thank Dr Kees Jalink for assistance with fluorescence spectroscopy. C.R.B. is supported by KWF grant 2005-3368.
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de Jong, A., Schuurman, K.G., Rodenko, B., Ovaa, H., Berkers, C.R. (2012). Fluorescence-Based Proteasome Activity Profiling. In: Drewes, G., Bantscheff, M. (eds) Chemical Proteomics. Methods in Molecular Biology, vol 803. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-364-6_13
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DOI: https://doi.org/10.1007/978-1-61779-364-6_13
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