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 Rho (refer to optional step C in protocol). The lysates were then incubated with glutathione resin and GST-Rhotekin-RBD (lanes 2 and 3). GTPγS-treated lysate was also incubated without GST-Rhotekin-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 Rho Rabbit Antibody. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
|Product Includes||Quantity (with Count)||Storage Temp|
|GST-Rhotekin-RBD||3 x 4 mg||-80°C|
|GDP||1 x 50 µl||-80°C|
|GTP γS||1 x 50 µl||-80°C|
|Rho Rabbit Antibody||1 x 75 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||4°C|
GTPγS: Store at -80°CGDP: Store at -80°CGST-Rhotekin-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°CRho Rabbit Antibody: Store at -20°C
The Active Rho Detection Kit provides all reagents necessary for measuring activation of Rho GTPase in the cell. GST-Rhotekin-RBD fusion protein is used to bind the activated form of GTP-bound Rho, which can then be immunoprecipitated with glutathione resin. Rho activation levels are then determined by western blot using a Rho Rabbit Antibody.
Active Rho Detection Kit detects endogenous levels of GTP-bound (active) Rho 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).
Rho family small GTPases, including Rho, Rac, and cdc42, act as molecular switches, regulating processes such as cell migration, adhesion, proliferation, and differentiation. They are activated by guanine nucleotide exchange factors (GEFs), which catalyze the exchange of bound GDP for GTP, and inhibited by GTPase activating proteins (GAPs), which catalyze the hydrolysis of GTP to GDP. A third level of regulation is provided by the stoichiometric binding of Rho GDP dissociation inhibitor (RhoGDI) (5). RhoA, RhoB and RhoC are highly homologous, but appear to have divergent biological functions. Carboxy-terminal modifications and differences in subcellular localization allow these three proteins to respond to and act on distinct signaling molecules (6,7).
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