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This protocol describes procedures to assay for GTP-bound (active) form of small GTPases of the Rho superfamily in human brain cancer (glioma) cell lines but can also be applied to cells or tissues of other origins. The principle of assay is based on the property of these active GTPases to interact with their specific effectors (e.g. Rhotekin for RhoA; p21-activated kinase (PAK) for Rac1 and Cdc42. In essence, Rhotekin or PAK1 are expressed in the form of GST-fusion protein to pull down the corresponding active GTPases.

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RhoGTPase Activation Assay

Biochemistry > Protein > Activity
Author: Alan Yiu Wah Lee
Alan Yiu Wah LeeAffiliation: Department of Physiology, Yong Loo Lin School of Medicine, Neurobiology/Ageing Programme, Life Sciences Institute, National University of Singapore (NUS), Singapore, Singapore
For correspondence: yiu_wah_lee@nuhs.edu.sg
Bio-protocol author page: a119
Vol 2, Iss 19, 10/5/2012, 3775 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.269

[Abstract] This protocol describes procedures to assay for GTP-bound (active) form of small GTPases of the Rho superfamily in human brain cancer (glioma) cell lines but can also be applied to cells or tissues of other origins. The principle of assay is based on the property of these active GTPases to interact with their specific effectors (e.g. Rhotekin for RhoA; p21-activated kinase (PAK) for Rac1 and Cdc42. In essence, Rhotekin or PAK1 are expressed in the form of GST-fusion protein to pull down the corresponding active GTPases.

Materials and Reagents

  1. Complete Protease Inhibitor Cocktail, EDTA-free (Roche Diagnostics GmbH, catalog number: 11873580001), prepared as 25x stock according to manufacturer’s instructions
  2. GST-Rhotekin (Millipore/Upstate, catalog number: 14-662)
  3. GST-PAK1 (in-house)
  4. anti-RhoA antibody (1:200) (Santa Cruz, catalog number: sc-418)
  5. anti-Rac1 antibody (1:200) (Santa Cruz, catalog number: sc-95)
  6. anti-Cdc42 antibody (1:500) (Santa Cruz, catalog number: sc-87)
  7. anti-GST antibody (1:2,000) (Santa Cruz, catalog number: sc-138)
  8. Glutathione (GSH) sepharose 4B (GE Healthcare Life Sciences, catalog number: 17-0756-01)
  9. Dithiothreitol (DTT) (Bio-Rad Laboratories, catalog number: 161-0611)
  10. Triton X-100 (Bio-Rad Laboratories, catalog number: 161-0407)
  11. Phosphate-buffered saline (PBS)
  12. Tris-HCl (pH 7.4)
  13. NaCl
  14. EDTA
  15. NP40
  16. Na2P2O7
  17. NaF
  18. Na3VO4
  19. Sample loading buffer
  20. 10% or 12% SDS-PAGE gel
  21. GST pull-down core buffer (see Recipes) 
  22. RIPA buffer (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, catalog number: 5415R)
  2. Nutating mixer (Labnet International)
  3. 1.5 ml Eppendorf tubes
  4. Heat block

Procedure

  1. Lyse cell samples by incubating in RIPA buffer supplemented with 1% Triton X-100 at 4 °C for 2 h. Centrifuge at 18,000 x g for 10 min. at 4 °C and collect the cleared crude cell lysate.
  2. Transfer 15 μl of GSH-sepharose into 100 μl of GST pull-down core buffer in a 1.5 ml Eppendorf tube. Mix well and spin at 835 x g for 1 min. at room temperature. Decant the supernatant and save the washed GSH-sepharose for pre-clearing the samples (step 3).
  3. Pre-clear 100 μg of crude cell lysates by adding into 300 μl of GST pull-down buffer (prepared by supplementing core buffer with 0.1% Triton X-100, 1 mM DTT, and 1x protease inhibitors) containing the washed GSH-sepharose (prepared in step 2). Incubate with agitation at 4 °C for 1 h.
    Note: If multiple samples, first bring up 100 μg lysate to equal volume with corresponding lysis buffer to ensure equal input.
  4. Centrifuge at 835 x g for 1 min at 4 °C. Transfer supernatant to a new 1.5 ml tube without disturbing the sepharose. Save an aliquot of the pre-cleared lysate as “sample input”.
  5. Add to the supernatant 30 μl glutathione beads bound with 30 μg GST-Rhotekin (for RhoA-GTP assay) or GST-PAK1 (for Rac1- and Cdc42-GTP assays). A “GST only” control is similarly prepared by adding GST pre-bound sepharose. Mix well and incubate at 4 °C for 2 to 3 h with rocking.
  6. Centrifuge at 835 x g for 1 min at 4 °C to pellet the sepharose beads.
  7. Aspirate the supernatant (optional: Save supernatant for quality control of pull-down reaction).
  8. Wash the pelleted sepharose beads by adding 300 μl 1x PBS at 4 °C.
  9. Spin at 835 x g for 1 min at 4 °C and aspirate supernatant (Optional: Save supernatant as “first wash” for quality control of washing step).
  10. Repeat washing. Save supernatant as “second wash” (Optional).
  11. Boil the beads in sample loading buffer for 5 min. and spin to harvest the pull-down proteins.
  12. Resolve the eluted proteins and an aliquot of cell/tissue lysates (sample input) by SDS-PAGE (10% or 12% gel). An aliquot of the “supernatant”, “first wash” and “second wash” collected above can also be included for quality control.
  13. Western blot analysis is then performed using anti-RhoA, Rac1, or Cdc42 antibodies to detect active (pull-down) and total (input) GTPases. The blot is also probed with anti-GST antibody to confirm successful GST pull down at comparable efficiency across samples.
    Note: Both Rhotekin and PAK1 preferentially bind to active (GTP-bound form) RhoA and Rac1/Cdc42, respectively. Quality control of these reagents can be performed by incubating GST-Rhotekin (or GST-PAK1) with recombinant RhoA (or Rac1/Cdc42) that has been preloaded with GDP or nonhydrolyzable GTP-γS.

Recipes

  1. GST pull-down core buffer
    20 mM Tris-HCl (pH 7.4)
    150 mM NaCl
    1 mM EDTA
  2. RIPA buffer
    50 mM Tris-HCl (pH 7.4)
    150 mM NaCl
    1% NP40
    1 mM Na2P2O7
    1 mM NaF
    1 mM EDTA
    2 mM Na3VO4

Acknowledgments

This protocol is adapted from Li et al. (2012).

References

  1. Li, X., Law, J. W. and Lee, A. Y. (2012). Semaphorin 5A and plexin-B3 regulate human glioma cell motility and morphology through Rac1 and the actin cytoskeleton. Oncogene 31(5): 595-610.


How to cite this protocol: Lee, A. Y. (2012). RhoGTPase Activation Assay. Bio-protocol 2(19): e269. DOI: 10.21769/BioProtoc.269; Full Text



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