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Scalable In Vitro Proteasome Activity Assay

  • Amit Kumar Singh Gautam
  • Kirby Martinez-Fonts
  • Andreas MatouschekEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1844)

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.

Key words

Proteasome Ubiquitin-proteasome system High-throughput degradation assay 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Amit Kumar Singh Gautam
    • 1
  • Kirby Martinez-Fonts
    • 1
  • Andreas Matouschek
    • 1
    Email author
  1. 1.Department of Molecular BiosciencesThe University of Texas at AustinAustinUSA

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