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Rho GTPases pp 253-270 | Cite as

High-Throughput Flow Cytometry Bead-Based Multiplex Assay for Identification of Rho GTPase Inhibitors

  • Zurab Surviladze
  • Susan M. Young
  • Larry A. Sklar
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 827)

Abstract

Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions, their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly.

Herein, we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out.

This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases.

Key words

Rho GTPases Cdc42 Bead-based multiplex assay Fluorescent GTP binding Screen Flow cytometry 

Notes

Acknowledgments

This work was supported by NIH grants U54 MH074425 and U54 MH084690.

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Zurab Surviladze
    • 1
    • 2
  • Susan M. Young
    • 1
    • 2
  • Larry A. Sklar
    • 1
    • 3
  1. 1.New Mexico Molecular Libraries Screening CenterAlbuquerqueUSA
  2. 2.Cancer Research and Treatment CenterUniversity of New Mexico School of MedicineAlbuquerqueUSA
  3. 3.Department of Pathology, Cancer Research and Treatment CenterUniversity of New Mexico School of MedicineAlbuquerqueUSA

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