RhoGTPase Activation Assay

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.