Rho GTPase Activation Assays

Stéphanie Pellegrin1, Harry Mellor1

1 Mammalian Cell Biology Laboratory, Department of Biochemistry, School of Medical Sciences, University of Bristol, United Kingdom
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.8
DOI:  10.1002/0471143030.cb1408s38
Online Posting Date:  March, 2008
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Abstract

The Rho GTPase family of signaling proteins controls a wide range of highly dynamic cellular processes. Activation of Rho GTPases can be investigated and quantified in cell extracts using so‐called pull‐down assays. Proteins that bind specifically to the activated form of the Rho GTPase are used to capture it onto a bead support. Western blotting of the captured samples with specific antibodies then allows for quantification of the level of Rho GTPase activation in the sample. This unit describes the techniques for preparing the reagents required for assays of RhoA, Rac, and Cdc42 and gives practical tips for the successful application of the assay in a range of situations. Curr. Protoc. Cell Biol. 38:14.8.1‐14.8.19. © 2008 by John Wiley & Sons, Inc.

Keywords: Rho GTPase; cytoskeleton; cell signaling

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Rac1 Activation Assays on Hela Cells
  • Support Protocol 1: Making GST‐RBD Beads for Activation Assays
  • Alternate Protocol 1: RhoA Activation Assays
  • Alternate Protocol 2: Loading Rac1 or Cdc42 with Nucleotide
  • Alternate Protocol 3: Rac1 Activation Assay on Nonadherent Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Rac1 Activation Assays on Hela Cells

  Materials
  • HeLa cells: 70% confluent 10‐cm plates (two plates per experiment)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 100 µg/ml epidermal growth factor (EGF; Calbiochem); store aliquots at −80°C
  • DMEM/0.1% (w/v) fatty‐acid‐free bovine serum albumin (BSA)
  • Tris‐buffered saline (TBS): 50 mM Tris·Cl (pH 7.6; see appendix 2A)/140 mM NaCl, ice cold
  • Lysis buffer B, ice cold (see recipe)
  • 4× SDS‐PAGE sample buffer (see recipe)
  • GST‐PAK‐CRIB beads in Tris wash buffer A/10% (v/v) glycerol ( protocol 2)
  • Tris wash buffer B (see recipe), ice cold
  • 2× SDS‐PAGE sample buffer (see recipe)
  • SDS‐PAGE gel (see unit 6.1)
  • Refrigerated centrifuge, 4°C
  • Heating block, 95°C
  • 0.5‐ to 20‐µl GELoader tips (Eppendorf)
  • Additional reagents and equipment for performing SDS‐PAGE (unit 6.1) and immunoblotting (unit 6.2)

Support Protocol 1: Making GST‐RBD Beads for Activation Assays

  Materials
  • LB broth (unit 20.2)
  • 100 mg/ml ampicillin stock solution in water; store small aliquots at −20°C
  • 34 mg/ml chloramphenicol stock solution in ethanol; store small aliquots at −20°C, protected from light
  • E. coli: e. g., BL21(DE3)pLysS (Stratagene)
  • GST‐RBD construct (obtained from the authors or self‐constructed; see Figure )
  • 1 M isopropyl‐β‐D‐thiogalactopyranoside (IPTG); store small aliquots at −20°C
  • Glutathione‐Sepharose 4B beads (Amersham)
  • Lysis buffer A (see recipe), ice cold
  • Tris wash buffer A (see recipe), ice cold
  • Tris wash buffer A (see recipe)/10% (v/v) glycerol, ice cold
  • 4× SDS‐PAGE sample buffer (see recipe)
  • 12% SDS‐PAGE gel (see unit 6.1)
  • Bovine serum albumin (BSA)
  • Coomassie blue staining solution (unit 6.1)
  • Shaking incubator, 37°C
  • 2‐liter flasks
  • Refrigerated centrifuge, 4°C
  • 30‐ml centrifuge tubes, capable of withstanding 17000 × g for 30 min (e. g., Oak Ridge), prechilled
  • Sonicator
  • 15‐ and 50‐ml polypropylene tubes (e. g., Falcon), prechilled
  • End‐over‐end rotator, 4°C
  • Heating block, 95°C
  • Microcentrifuge tubes, prechilled
  • Additional reagents and equipment for performing SDS‐PAGE (unit 6.1)

Alternate Protocol 1: RhoA Activation Assays

  Materials
  • 10‐cm plates of cells (two plates for each experiment)
  • Tris‐buffered saline (TBS): 50 mM Tris·Cl (pH 7.6; see appendix 2A)/140 mM NaCl, ice cold
  • Lysis buffer D (see recipe), ice cold
  • 4× SDS‐PAGE sample buffer (see recipe)
  • 10 mM GTPγS (Calbiochem)/20 mM HEPES, pH 7.4; store aliquots at −20°C
  • 100 mM GDP (Sigma)/20 mM HEPES, pH 7.4; store aliquots at −20°C
  • 0.5 M EDTA, pH 8.0 (see appendix 2A)
  • 1 M MgCl 2
  • GST‐RBD beads in Tris wash buffer A/10% glycerol ( protocol 2)
  • Tris wash buffer C (see recipe), ice cold
  • 2× SDS‐PAGE sample buffer (see recipe), 95°C
  • SDS‐PAGE gel (see unit 6.1)
  • Refrigerated centrifuge, 4°C
  • Shaking incubator, 30°C
  • Heating block, 95°C
  • End‐over‐end rotator, 4°C
  • 0.5‐ to 20‐µl GELoader tips (Eppendorf)
  • Additional reagents and equipment for determining protein concentration ( appendix 3B or 3H) and performing SDS‐PAGE (unit 6.1) and immunoblotting (unit 6.2)

Alternate Protocol 2: Loading Rac1 or Cdc42 with Nucleotide

  Materials
  • Cultures of cells in suspension
  • RPMI/0.1% (w/v) fatty‐acid‐free BSA
  • RPMI/0.1% (w/v) fatty‐acid‐free BSA/20 mM HEPES, pH 7.4
  • SDF1α (R&D #350‐NS)
  • 2× lysis buffer (see recipe), ice cold
  • GST‐PAK‐CRIB beads in Tris wash buffer A/10% (v/v) glycerol ( protocol 2)
  • 4× SDS‐PAGE sample buffer (see recipe)
  • 2× SDS‐PAGE sample buffer (see recipe)
  • SDS‐PAGE gel (see unit 6.1)
  • Refrigerated centrifuge, 8°C
  • 0.5‐ to 20‐µl GELoader tips (Eppendorf)
  • Heating block, 95°C
  • Additional reagents and equipment for performing SDS‐PAGE (unit 6.1) and immunoblotting (unit 6.2)
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Figures

Videos

Literature Cited

Literature Cited
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