Thermo Scientific™ Active Rap1 Pull-Down and Detection Kit

Description

• Measure activation of small GTPases by isolating them via their specific downstream effectors with Thermo Scientific™ Pierce Active GTPase Pull-Down and Detection Kits
• 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