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 Rap1 (refer to optional step C in protocol). The lysates were then incubated with glutathione resin and GST-RalGDS-RBD (lanes 2 and 3). GTPγS-treated lysate was also incubated without GST-RalGDS-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 Rap1 Rabbit Antibody. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody. Detection was performed using 1X LumiGLO® reagent and peroxide (20X LumiGLO® Reagent and 20X Peroxide #7003), followed by exposure to x-ray film.
|Product Includes||Quantity (with Count)||Storage Temp|
|GST-RalGDS-RBD||1 x 600 µg||-80°C|
|GDP||1 x 50 µl||-80°C|
|GTP γS||1 x 50 µl||-80°C|
|Rap1 Rabbit mAb||1 x 50 µl||-20°C|
|Glutathione Resin||1 x 3 ml||4°C|
|SDS Sample Buffer||1 x 1.5 ml||4°C|
|Lysis/Binding/Wash Buffer||1 x 100 ml||4°C|
|Spin Cup and Collection Tubes||1 x 30 ea||RT|
GTPγS: Store at -80°CGDP: Store at -80°CGST-RalGDS-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°CRap1 Rabbit mAb: Store at 4°C
The Active Rap1 Detection Kit provides all reagents necessary for measuring activation of Rap1 GTPase in the cell. GST-RalGDS-RBD fusion protein is used to bind the activated form of GTP-bound Rap1, which can then be immunoprecipitated with glutathione resin. Rap1 activation levels are then determined by western blot using a Rap1 Rabbit Antibody.
Active Rap1 Detection Kit detects endogenous levels of GTP-bound (active) Rap1 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).
Rap1 and Rap2 belong to the Ras subfamily of small GTPases and are activated by a wide variety of stimuli through integrins, receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCR), death domain associated receptors (DD-R), and ion channels (5,6). Like other small GTPases, Rap activity is stimulated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase activating proteins (GAPs). A wide variety of Rap GEFs have been identified: C3G connects Rap1 with RTKs through adaptor proteins such as Crk, Epacs (or cAMP-GEFs) transmit signals from cAMP, and CD-GEFs (or CalDAG-GEFs) convey signals from either or both Ca2+ and DAG (5). Rap1 primarily regulates multiple integrin-dependent processes such as morphogenesis, cell-cell adhesion, hematopoiesis, leukocyte migration and tumor invasion (5,6). Rap1 may also regulate proliferation, differentiation and survival through downstream effectors including B-Raf, PI3K, RalGEF and phospholipases (PLCs) (5-8). Rap1 and Rap2 are not fuctionally redundant as they perform overlapping but distinct functions (9). Recent research indicates that Rap2 regulates Dsh subcellular localization and is required for Wnt signaling in early development (10).
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