Description
Storage
GTPγS: Store at -80°C GDP: Store at -80°C GST-Raf1-RBD: Store at -80°C Lysis/Binding/Wash Buffer: Store at 4°C Glutathione Resin: Store at 4°C SDS Sample Buffer: Store at 4°C Spin Cup and Collection Tubes: Store at RT Ras Mouse mAb: Store at 4°C
Protocol
Available protocols: Functional
Specificity / Sensitivity
Active Ras Detection Kit detects endogenous levels of GTP-bound (active) Ras as shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species. The Ras Mouse Monoclonal Antibody #8832 included in the kit will detect H, K, and N Ras and cross-reacts with human, mouse, and rat Ras.
Species Reactivity: Human, Mouse
Background
The Ras superfamily of small GTP-binding proteins (G proteins) comprise a large class of proteins (over 150 members) that can be classified into at least five families based on their sequence and functional similarities: Ras, Rho, Rab, Arf, and Ran (1-3). These small G proteins have both GDP/GTP-binding and GTPase activities and function as binary switches in diverse cellular and developmental events that include cell cycle progression, cell survival, actin cytoskeletal organization, cell polarity and movement, and vesicular and nuclear transport (1). An upstream signal stimulates the dissociation of GDP from the GDP-bound form (inactive), which leads to the binding of GTP and formation of the GTP-bound form (active). The activated G protein then goes through a conformational change in its downstream effector-binding region, leading to the binding and regulation of downstream effectors. This activation can be switched off by the intrinsic GTPase activity, which hydrolyzes GTP to GDP and releases the downstream effectors. These intrinsic guanine nucleotide exchange and GTP hydrolysis activities of Ras superfamily proteins are also regulated by guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound form and GTPase activating proteins (GAPs) that return the GTPase to its GDP-bound inactive form (4). The 21 kDa guanine-nucleotide binding proteins (K-Ras, H-Ras, and N-Ras) cycle between active (GTP-bound) and inactive (GDP-bound) forms (5). Receptor tyrosine kinases and G-protein-coupled receptors activate Ras, which then stimulates the Raf-MEK-MAPK pathway (6-8). GAP proteins normally facilitate the inactivation of Ras. However, in 30% of human tumors, point mutations in Ras prevent the GAP-mediated inhibition of this pathway (9). The most common oncogenic Ras mutation found in tumors is Gly12 to Asp (G12D), which prevents Ras inactivation, possibly by increasing the overall rigidity of the protein (9,10).
Alternate Names
11521; 11522; 11523; 11524; 11525; 11526; 11860; 11871; 8784; 8832; GTPase; raf; ras
