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.

Western blot result for Active GTPase Pull-Down and Detection Kits. alt=

Detection of active GTPases using Ras, Cdc42, Rac1, Rho, and Rap1 Pull-Down Kits (above right). NIH3T3 cell lysates were treated with GTPgS (activator) or GDP (inactivator) and then purified using respective GTPase Kits. Half of each eluate (25 µl) and 20 µg of lysate were analyzed by Western blotting using specific antibodies supplied in the respective kits. Only active GTPases were detected (see GDP lanes).

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.

Product #	Description	Pkg. Size
26185	Active Arf1 Pull-Down and Detection Kit Contains GST-GGA3-PBD fusion protein.	Kit
26186	Active Arf6 Pull-Down and Detection Kit Contains GST-GGA3-PBD fusion protein.	Kit
89857	Active Cdc42 Pull-Down and Detection Kit Contains GST-Pak1-PBD fusion protein.	Kit
89856	Active Rac1 Pull-Down and Detection Kit Contains GST-Pak1-PBD fusion protein.	Kit
26187	Active RalA Pull-Down and Detection Kit Contains GST-RalBP1-PBD fusion protein.	Kit
89872	Active Rap1 Pull-Down and Detection Kit Contains GST-RalGDS-RBD fusion protein.	Kit
89855	Active Ras Pull-Down and Detection Kit Contains GST-Raf1-RBD fusion protein.	Kit
89854	Active Rho Pull-Down and Detection Kit Contains GST-Rhotekin-RBD fusion protein.	kit