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.
Bioyears 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.
Bioyears 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.
1. Anti active Ras, Mouse Monoclonal Antibody (Catalog No. 26909): One vial – 22 µL (1 mg/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, 750mM 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.
Store all kit components at 4ºC until their expiration dates.