Complete kits to activate, purify and detect specific small GTPases, including Arf1, Arf6, Cdc42, Rac1, RalA, Rap1, Ras and Rho.

The Thermo Scientific Pierce Active GTPase Pull-down and Detection Kits measure activation of small GTPases by isolating them via their specific downstream effectors. Kits specific for Arf1, Arf6, Cdc42, Rac1, RalA, Rap1, Ras and Rho GTPases are offered. The respective binding domain of the downstream effector for each small GTPase is expressed as a GST-fusion protein which, when immobilized on a resin, is used to pull down the active (i.e., GTP-bound) GTPase. The pull-down affinity purification step is easily performed in convenient microcentrifuge spin columns. After a simple wash step, the bound GTPase is recovered by eluting the GST-fusion protein from the glutathione resin using SDS-PAGE sample loading buffer. The purified GTPase is then detected by Western blot using a specific antibody supplied in the each kit.

Highlights:

• High sensitivity and accuracy – optimized reagents, specific antibodies and Western blot procedure ensure accurate controls and quantitative results
• Convenience – no need to express and purify your own GST-PBD or RBD fusion proteins or use expensive antibody-based immunoprecipitation affinity resins
• Speed – simultaneous incubation of cell lysate with GST-PBD or RBD and the glutathione resin directly in a high-performance microcentrifuge spin column
• Ease-of-use – reagents and pull-down conditions have been optimized for immediate success, even for first-time users
• Efficiency – the microcentrifuge spin columns and collection tubes provide efficient separation of liquid and resin, preventing sample loss
• Complete – kits include reagents for affinity purification as well as controls, a compatible cell lysis buffer and specific primary antibody for final Western blot detection

GST fusion proteins of active GTPase binding domains:

• Raf1 Ras-binding domain (Raf1-RBD) binds active Ras
• Pak1 p21-binding domain (Pak1-PBD) binds active Rac1 and Cdc42
• Rhotekin Rho-binding domain (Rhotekin-RBD) binds active Rho
• RalGDS Rap1-binding domain (RalGDS-RBD) binds active Rap1
• GGA3 protein-binding domain (GGA3-PBD) binds active Arf1 and Arf6
• Ral binding protein 1 (RalBP1) binds active RalA

Product Details:

Each GTPase Kit includes GST-fusion protein of the specific binding domain, GTPgS and GDP activators, glutathione agarose resin, lysis/binding/washing buffer, primary antibody, 2X SDS sample buffer, spin columns and collection tubes. Each kit contains sufficient components for 30 pull-down assays. The kits are optimized for Western blot detection with an HRP-conjugated secondary antibody (Product # 31460-rabbit or 31430-mouse) using Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Product # 34080). Monomeric p21 GTP-binding proteins (small GTPases) serve as molecular switches in regulating a wide range of essential biochemical pathways in eukaryotic cells. The families of Ras and Rho (including Cdc42, Rac1, Rho and Rap1) are of special interest as they influence the cell’s response to the changing environment. Research results have indicated that these proteins regulate numerous cell functions such as proliferation, differentiation, transformation, apoptosis, migration, actin cytoskeleton reorganization, and cell cycle progression. Like other G-proteins, small GTPases cycle between an inactive, GDP-bound state and an active, GTP-bound state. To validate the specificity and function of the purification method, cell lysates were treated with GTPgS to activate the endogenous Ras, Cdc42, Rac1, Rho, or Rap1 or treated with GDP to inactivate these small GTPases. Strong signals for the specific targets were detected by Western blotting using GTPgS-treated cell lysate, while very little or no signal was detected when GDP-treated cell lysate was used (see Figure). Furthermore, no signal was detected when GST alone was incubated with GTPgS-treated cell lysate (negative control; data not shown). Therefore, the Active GTPase Pull-down and Detection Kits specifically measure each active small GTPase.

Poster Presentation:

• Smith, S.M., Bomgarden, R.D., Deshpande, A., Farooqui, R. and Kaboord, B.J. (2008). Comparison of small GTPase activation over time after growth factor stimulation in 3T3 cells. Mol. Biol. Cell 19 (suppl), abstract 1243/B452.

General GTPase References:

1. Asha, H., et al. (1999). The Rap1 GTPase functions as a regulator of morphogenesis in vivo. EMBO J. 18, 605-615.
2. Bar-Sagi, D. and Hall, A. (2000). Ras and Rho GTPases: a family reunion. Cell 103, 227-238.
3. Taylor, S.J., et al. (2001). Nonradioactive determination of Ras-GTP levels using activated Ras interaction assay. Methods Enzymol. 333, 333-348.
4. Benard, V., et al. (1999). Characterization of Rac and Cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J. Biol. Chem. 274, 13198-13204.
5. Benard V. and Bokoch, G.M. (2002). Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol. 345, 349-359.
6. Cantor, S., et al. (1995). Identification and characterization of Ral-Binding Protein1, a potential downstream target of Ral GTPases. Mol Cell Biol. 15, 4578-84.
7. Charest, P. and Firtel, R. (2007). Big roles for small GTPases in the control of directed cell movement. Biochem J. 401, 377-90.
8. Dell'Angelica, E., et al. (2000). GGAs: A family of ADP ribosylation factor-binding proteins related to adaptors and associated with the golgi complex. J Cell Biol. 149, 81-93.
9. Draveling, C., et al. (2001). SwellGel: an affinity chromatography technology for high capacity and high throughput purification of recombinant-tagged proteins. Protein Expression Purif. 22, 359-366.
10. D'Souza-Schorey, C. and Chavrier, P. (2006). ARF proteins: Roles in membrane traffic and beyond. Nat Rev Mol Cell Biol. 7, 347-58.
11. Feig, L. (2003). Ral-GTPases: Approaching their 15 minutes of fame. Trends Cell Biol. 13, 419-25.
12. Foschi, M., et al. (1997). Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase. EMBO J. 16, 6439-6451.
13. Knox, A.L. and Brown, N.H. (2002). Rap1 GTPase regulation of adherens junction positioning and cell adhesion. Science 295, 1285-1288.
14. Reid, T., et al. (1996). Rhotekin, a new putative target for Rho bearing homology to a serine/ threonine kinase, PKN, and rhophilin in the Rho-binding domain. J. Biol. Chem. 271, 13556-13560.
15. Ren, X.-D., et al. (1999). Regulation of the small FTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18, 578-585.
16. Ren, X.-D. and Schwartz, M.A. (2000). Determination of GTP loading on Rho. Methods Enzymol. 325, 264-272.
17. Van Triest, M., et al. (2001). Measurement of GTP-bound Ras-like GTPases by activation-specific probes. Methods Enzymol. 333, 343-348.
18. Williams, D., et al. (2008). Rho GTPases and regulation of hematopoietic stem cell localization. Methods Enzymol. 439, 365-393.
19. Yoon, H.Y., et al. (2005). In vitro assays of Arf1 interaction with GGA proteins. Methods Enzymol. 404, 316-332.
20. Zwartkruis, F.J.T. and Bos, J.L. (1999). Ras and Rap1: two highly related small GTPases with distinct function. Exp. Cell Res. 253, 157-165.

Kit Contents:

GTPase Pull-down and Detection Kits contain sufficient materials to perform 30 pull-down assays from a 0.5 to 1mg cell lysate sample. All kits have the following component structure:

• GST Fusion Protein of Specific Binding Domain, 1 vial
• Glutathione Agarose Resin, 3mL
• GTPgammaS (100X), 50µL
• GDP (100X), 50µL
• Lysis/Binding/Wash Buffer, 100mL
• GTPase-Specific Primary Antibody, 1 vial
• SDS-PAGE Sample Loading Buffer (2X), 1.5mL
• Spin Columns, 30 columns
• Collection Tubes, 90 tubes