Materials and Reagents

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

Equipment

Centrifuge (Eppendorf, catalog number: 5415R )
Nutating mixer (Labnet International)
1.5 ml Eppendorf tubes
Heat block

Procedure

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.
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).
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.
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”.
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.
Centrifuge at 835 x g for 1 min at 4 °C to pellet the sepharose beads.
Aspirate the supernatant (optional: Save supernatant for quality control of pull-down reaction).
Wash the pelleted sepharose beads by adding 300 μl 1x PBS at 4 °C.
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).
Repeat washing. Save supernatant as “second wash” (Optional).
Boil the beads in sample loading buffer for 5 min. and spin to harvest the pull-down proteins.
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.
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

GST pull-down core buffer
20 mM Tris-HCl (pH 7.4)
150 mM NaCl
1 mM EDTA
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

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.