Ras Pull-Down Pull-Down Assay

Ras Pull-Down Activation Assay Kit

Cat. # TD-81101

Background

Small GTPases are a super-family of cellular signaling regulators. Ras belongs to the Ras sub-family of GTPases that regulate cell growth, cell motility, and gene transcription. GTP binding increases the activity of Ras, and the hydrolysis of GTP to GDP renders it inactive.
Currently the activation of Ras proteins is assayed with the binding of GTP-bound Ras to the Ras-binding domain (RBD) of Raf protein kinase. This method is based on the ob/servation that the active, GTP-bound Ras could bind to the RBD of Raf. 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 RBD to Ras-GTP.
The Ras Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Ras-GTP, but not Ras-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 Ras Activation Assay Kit uses configuration-specific anti-Ras-GTP Mouse monoclonal antibody to measure Ras-GTP levels in cell extracts or in vitro GTPγS loading Ras activation assays. Anti-Ras-GTP mouse monoclonal antibody is first incubated with cell lysates containing Ras-GTP. Next, the GTP-bound Ras is pulled down by protein A/G agarose. Finally, the precipitated Ras-GTP is detected through immunoblot analysis using Anti-Ras Rabbit Polyclonal Antibody.
The anti-Ras-GTP monoclonal antibody can also be used to monitor the activation of Ras in cells and in tissues by immunohistochemistry.