Cdc42 Pull-Down Activation Assay Kit

Cat. # TD-80701

Background

Small GTPases are a super-family of cellular signaling regulators. Cdc42 belongs to the Rho sub-family of GTPases that regulate cell motility, cell division, and gene transcription. GTP binding increases the activity of Cdc42, and the hydrolysis of GTP to GDP renders it inactive.

Currently the activation of Cdc42 proteins is assayed with the binding of GTP-bound Cdc42 to the p21-binding domain (PBD) of p21-activated protein kinase (PAK). This method is based on the observation that the active, GTP-bound Cdc42 (and 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 Cdc42-GTP.

The Cdc42 Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Cdc42-GTP, but not Cdc42-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.

Assay Principle

The Cdc42 Activation Assay Kit uses configuration-specific anti-Cdc42-GTP Mouse monoclonal antibody to measure Cdc42-GTP levels in cell extracts or in vitro GTPγS loading Cdc42 activation assays. Anti-Cdc42-GTP mouse monoclonal antibody is first incubated with cell lysates containing Cdc42-GTP. Next, the GTP-bound Cdc42 is pulled down by protein A/G agarose. Finally, the precipitated Cdc42-GTP is detected through immunoblot analysis using Anti-Cdc42 Rabbit Polyclonal Antibody.

The anti-Cdc42-GTP monoclonal antibody can also be used to monitor the activation of Cdc42 in cells and in tissues by immunohistochemistry.