Description
Quantity: 30 reactions
Unit Size: Each
The 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. When immobilized on a resin, this fusion protein is used to pull down the active (i.e., GTP-bound) GTPase. The pull-down affinity purification step is easily and conveniently performed using 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 each kit. Each GTPase Kit includes GST-fusion protein of the specific binding domain, GTPγS and GDP activators, glutathione agarose resin, lysis-binding-wash 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 (Part No. 31460, rabbit; Part No. 31430, mouse) using Thermo Scientific SuperSignal™ West Pico Chemiluminescent Substrate (Part No. 34080). 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 Includes: GST fusion protein of specific binding domain, glutathione agarose resin, GTPγS and GDP (100X), lysis-binding-wash buffer, GTPase-specific primary antibody, SDS-PAGE sample loading buffer (2X), spin columns and collection tubes
