Configuration-specific Monoclonal Antibody Based

Ras Activation Assay Kit

Catalog Number：CR-W81101

20 assays

Product Description

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 observation 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.

NewEast Biosciences 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.

These anti-Ras-GTP monoclonal antibody can also be used to monitor the activation of Ras in cells and

in tissues by immunohistochemistry.

NewEast Biosciences Ras Activation Assay Kit provides a simple and fast method to monitor the

activation of Ras. Each kit provides sufficient quantities to perform 20 assays.

Assay Principle

NewEast Biosciences Ras Activation Assay Kit bases on the configuration-specific anti-Ras-GTP

monoclonal antibody to measure the active Ras-GTP levels, either from cell extracts or from in vitro

GTPγS loading Ras activation assays. Briefly, anti-active Ras mouse monoclonal antibody will be

incubated with cell lysates containing Ras-GTP. The bound active Ras will then be pulled down by

protein A/G agarose. The precipitated active Ras will be detected by immunoblot analysis using anti-Ras

rabbit polyclonal antibody.

Kit Components

1. Anti-active Ras, Mouse Monoclonal Antibody (Catalog No. 26909): One vial – 22 µL (1mg/ml) in PBS, pH 7.4, containing 50% glycerol and 0.05% sodium azide. This antibody

specifically recognizes Ras-GTP from all vertebrates.

2. Protein A/G Agarose (Catalog No. 30301): One vial – 400 µL of 50% slurry.

3. 5X Assay/Lysis Buffer (Catalog No. 30303): 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-Ras, Rabbit polyclonal Antibody (Catalog No. 21021): One vial – 22 µL (1 mg/ml) in

PBS, pH 7.4, contained 50% glycerol.

5. 100 X GTPγS (Catalog No. 30302): One vial –100 µl at 10 mM, use 5 µL of GTPγS for

GTP-labeling of 0.5 mL of cell lysate.

6. 100 X GDP (Catalog No. 30304): One vial –100 µl at 100 mM, use 5 µL of GDP for

GDP-labeling of 0.5 mL of cell lysate.

Storage

Store all kit components at 4ºC until their expiration dates.