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