Figure 2. The GTP-bound GTPase pull-down process can be divided into 3 steps as shown. Step 1: Mix sample, binding protein, and glutathione resin in the spin cup and incubate at 4ºC to allow GTP-bound GTPase binding to the glutathione resin through GST-linked binding protein. Step 2: Remove unbound proteins by centrifugation. Step 3: Elute glutathione resin-bound GTPase with SDS buffer. The eluted sample can then be analyzed by western blot.
Figure 1. NIH/3T3 cell lysates (500 µl at 1 mg/ml) were treated in vitro with GTPγS or GDP to activate or inactivate Ras (refer to optional step C in protocol). The lysates were then incubated with glutathione resin and GST-Raf1-RBD (lanes 2 and 3). GTPγS-treated lysate was also incubated without GST-Raf1-RBD in the presence of glutathione resin as a negative control (lane 4). Western blot analysis of cell lysate (20 µg, lane 1) or 20 µl of the eluted samples (lanes 2, 3, and 4) was performed using a Ras mouse mAb. Anti-mouse IgG, HRP-linked Antibody #7076 was used as the secondary antibody.
|Product Includes||Quantity (with Count)||Storage Temp|
|GTP γS||1 x 50 µl||-80°C|
|GDP||1 x 50 µl||-80°C|
|Glutathione Resin||1 x 3 ml||4°C|
|Lysis/Binding/Wash Buffer||1 x 100 ml||4°C|
|SDS Sample Buffer||1 x 1.5 ml||4°C|
|Spin Cup and Collection Tubes||1 x 30 ea||RT|
|GST-Raf1-RBD||1 x 2.4 mg||-80°C|
|Ras Mouse mAb||1 x 250 µl||4°C|
GTPγS: Store at -80°CGDP: Store at -80°CGST-Raf1-RBD: Store at -80°CLysis/Binding/Wash Buffer: Store at 4°CGlutathione Resin: Store at 4°CSDS Sample Buffer: Store at 4°CSpin Cup and Collection Tubes: Store at 4°CRas Mouse mAb: Store at 4°C
The Active Ras Detection Kit provides all reagents necessary for measuring activation of Ras GTPase in the cell. GST-Raf1-RBD fusion protein is used to bind the activated form of GTP-bound Ras, which can then be immunoprecipitated with glutathione resin. Ras activation levels are then determined in western using a Ras mouse mAb.
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 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).
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