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 Rac1 (refer to optional step C in protocol). The lysates were then incubated with glutathione resin and GST-PAK1-PBD (lanes 2 and 3). GTPγS-treated lysate was also incubated without GST-PAK1-PBD 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 Rac1 Mouse mAb. Anti-mouse IgG, HRP-linked Antibody #7076 was used as the secondary antibody.
|Product Includes||Quantity (with Count)||Storage Temp|
|GST-Human PAK1-PBD||1 x 600 µg||-20°C|
|Rac1 Mouse Antibody||1 x 50 µl||-20°C|
|GDP||1 x 50 µl||-80°C|
|GTP γS||1 x 50 µl||-80°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-Human PAK1-PBD: Store at -20°CRac1 Mouse mAb: Store at -20°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°C
NOTE: We recommend making 1 mg/ml lysate in 1X Lysis/Binding/Wash Buffer for the following steps.
Perform the following treatments, GTPγS (positive control) and GDP (negative control), to ensure the immunoprecipitation procedures are working properly. Use 500 µg of cell lysate for each treatment. For best results, aliquot GTPγS and GDP at first use to minimize freeze/thaw cycles.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
NOTE: CST recommends loading prestained molecular weight markers (#74124, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights. 4-20% acrylamide or Tris-glycine gel provides best separation.
NOTE: LumiGLO® substrate can be further diluted if signal response is too fast.
NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following LumiGLO® incubation and declines over the following 2 hr.
LumiGLO® is a registered trademark of Kirkegaard & Perry Laboratories.
posted June 2020
Protocol Id: 2045
The Active Rac1 Detection Kit provides all reagents necessary for measuring activation of Rac1 GTPase in the cell. GST-PAK1-PBD fusion protein is used to bind the activated form of GTP-bound Rac1, which can then be immunoprecipitated with glutathione resin. Rac1 activation levels are then determined by western blot using a Rac1 Mouse mAb.
Active Rac1 Detection Kit detects endogenous levels of GTP-bound (active) Rac1 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).
Rac and Cdc42 are members of the Rho-GTPase family. In mammals, Rac exists as three isoforms, Rac1, Rac2, and Rac3, which are highly similar in sequence. Rac1 and Cdc42, the most widely studied of this group, are ubiquitously expressed. Rac2 is expressed in cells of hematopoietic origin, and Rac3, while highly expressed in brain, is also found in many other tissues. Rac and Cdc42 play key signaling roles in cytoskeletal reorganization, membrane trafficking, transcriptional regulation, cell growth, and development (5). GTP binding stimulates the activity of Rac/Cdc42, and the hydrolysis of GTP to GDP through the protein's intrinsic GTPase activity, rendering it inactive. GTP hydrolysis is aided by GTPase activating proteins (GAPs), while exchange of GDP for GTP is facilitated by guanine nucleotide exchange factors (GEFs). Another level of regulation is achieved through the binding of RhoGDI, a guanine nucleotide dissociation inhibitor, which retains Rho family GTPases, including Rac and Cdc42, in their inactive GDP-bound state (6,7).
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