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Small-Molecule High-Throughput Screening Utilizing Xenopus Egg Extract

  • Matthew R. Broadus
  • P. Renee Yew
  • Stephen R. Hann
  • Ethan Lee
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1263)

Abstract

Screens for small-molecule modulators of biological pathways typically utilize cultured cell lines, purified proteins, or, recently, model organisms (e.g., zebrafish, Drosophila, C. elegans). Herein, we describe a method for using Xenopus laevis egg extract, a biologically active and highly tractable cell-free system that recapitulates a legion of complex chemical reactions found in intact cells. Specifically, we focus on the use of a luciferase-based fusion system to identify small-molecule modulators that affect protein turnover.

Key words

Xenopus egg extract Xenopus laevis Cell-free Small molecules High-throughput screening Protein turnover Protein degradation 

Notes

Acknowledgments

We thank Laurie Lee for critical reading of the manuscript. M.R.B. is supported by a National Cancer Institute training grant (T32 CA119925). E.L. is supported by the National Institutes of Health (R01GM081635 and R01GM103926). R.Y. is supported by Award Number 8UL1TR000149 from the National Center for Advancing Translational Sciences and the CTRC P30 Cancer Center Support Grant from the National Cancer Institute (CA054174). S.R.H. is supported by the National Cancer Institute (P50 CA095103).

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Matthew R. Broadus
    • 1
  • P. Renee Yew
    • 2
  • Stephen R. Hann
    • 1
  • Ethan Lee
    • 1
    • 3
    • 4
  1. 1.Department of Cell and Developmental BiologyVanderbilt University Medical CenterNashvilleUSA
  2. 2.Department of Molecular Medicine, Institute of BiotechnologyThe University of Texas Health Science Center at San AntonioSan AntonioUSA
  3. 3.Vanderbilt Institute of Chemical BiologyVanderbilt University Medical CenterNashvilleUSA
  4. 4.Vanderbilt-Ingram Cancer CenterVanderbilt University Medical CenterNashvilleUSA

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