Rac Pull-Down Activation Assay Kit

Cat. # TD-80501

Background

Small GTPases are a super-family of cellular signaling regulators. Rac belongs to the Rho sub-family of GTPases that regulate cell motility, cell division, and gene transcription. GTP binding increases the activity of Rac, and the hydrolysis of GTP to GDP renders it inactive.
Currently the activation of Rac proteins is assayed with the binding of GTP-bound Rac to the p21-binding domain (PBD) of p21-activated protein kinase (PAK). This method is based on the ob/servation that the active, GTP-bound Rac 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 Rac-GTP.
The Rac Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Rac-GTP, but not Rac-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.
These anti-Rac-GTP monoclonal antibody can also be used to monitor the activation of Rac in cells and in tissues by immunohistochemistry.

Assay Principle

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