Abstract
We developed a degradation assay based on fluorescent protein substrates that are efficiently recognized, unfolded, translocated, and hydrolyzed by the proteasome. The substrates consist of three components: a proteasome-binding tag, a folded domain, and an initiation region. All the components of the model substrate can be changed to modulate degradation, and the assay can be performed in parallel in 384-well plates. These properties allow the assay to be used to explore a wide range of experimental conditions and to screen proteasome modulators.
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Acknowledgment
This work was supported by U54 GM105816, R21 CA191664, R21 CA196456, and R01 GM124501 from the National Institutes of Health; RP140328 from the Cancer Prevention and Research Institute of Texas (CPRIT); and F-1817 from the Welch.
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Singh Gautam, A.K., Martinez-Fonts, K., Matouschek, A. (2018). Scalable In Vitro Proteasome Activity Assay. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_21
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