Small GTPases are a super-family of cellular signaling regulators. RhoA belongs to the Rho sub-family of GTPases that regulate cell motility, cell division, and gene transcription. GTP binding increases the activity of RhoA, and the hydrolysis of GTP to GDP renders it inactive.

Currently the activation of RhoA proteins is assayed with the binding of GTP-bound RhoA to the p21-binding domain (PBD) of p21-activated protein kinase (PAK). This method is based on the observation that the active, GTP-bound RhoA could bind to the PBD of PAK. However, the reproducibility of this method is poor. This is partially due to the relatively quick hydrolysis of GTP to GDP during the assay procedure, and the low binding affinity of PBD to RhoA-GTP.

The RhoA Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes RhoA-GTP, but not RhoA-GDP. Given the high affinity of monoclonal antibodies to their antigens, the activation assay could be performed in a much shorter time. This assay provides the reliable results with consistent reproducibility.

The anti-RhoA-GTP monoclonal antibody can also be used to monitor the activation of RhoA in cells and in tissues by immunohistochemistry.

The RhoA Activation Assay Kit uses configuration-specific anti-RhoA-GTP Mouse monoclonal antibody to measure RhoA-GTP levels in cell extracts or in vitro GTPγS loading RhoA activation assays. Anti-RhoA-GTP mouse monoclonal antibody is first incubated with cell lysates containing RhoA-GTP. Next, the GTP-bound RhoA is pulled down by protein A/G agarose. Finally, the precipitated RhoA-GTP is detected through immunoblot analysis using Anti-RhoA Rabbit Polyclonal Antibody.

1. Anti-RhoA-GTP Mouse Monoclonal Antibody (Cat. # 26904): One vial – 35 μL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes RhoA-GTP from all vertebrates.

2. Protein A/G Agarose (Cat. # 30301): One vial – 600 μL of 50% slurry.

3. 5X Assay/Lysis Buffer (Cat. # 30302): One bottle – 30 mL of 250 mM Tris-HCl, pH 8, 750 mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.

4. Anti-RhoA Rabbit Polyclonal Antibody (Cat. # 21017): One vial – 50 μL (1 mg/mL) in PBS, pH 7.4, contained 50% glycerol.

5. 100X GTPγS (Cat. # 30303): One vial – 50 μl at 10 mM, use 5 μL of GTPγS for  GTP-labeling of 0.5 mL of cell lysate.

6. 100X GDP (Cat. # 30304): One vial – 50 μl at 100 mM, use 5 μL of GDP for GDP-labeling of 0.5 mL of cell lysate.

7. HRP-Goat Anti-Rabbit IgG (Cat. #29002): 50 μL (0.4 mg/mL) in PBS, pH 7.4, contained 50% glycerol.