RhoA Activation Assay Kit

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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 RhoA-binding domain (RBD) of the RhoA effector protein, Rhotekin. This method is based on the observation that the active, GTP-bound RhoA could bind to the RBD of rhotekin. 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 RhoA-GTP. NewEast Biosciences 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. This anti-RhoA-GTP monoclonal antibody can also be used to monitor the activation of RhoA in cells and in tissues by immunohistochemistry. NewEast Biosciences RhoA Activation Assay Kit provides a simple and fast method to monitor the activation of RhoA. Each kit provides sufficient quantities to perform 20 assays. Assay Principle NewEast Biosciences RhoA Activation Assay Kit bases on the configuration-specific anti-RhoA-GTP monoclonal antibody to measure the active RhoA-GTP levels, either from cell extracts or from in vitro GTPγS loading RhoA activation assays. Briefly, anti-active RhoA mouse monoclonal antibody will be incubated with cell lysates containing RhoA-GTP. The bound active RhoA will then be pulled down by protein A/G agarose. The precipitated active RhoA will be detected by immunoblot analysis using anti-RhoA rabbit polyclonal antibody.

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