Ras Activation ELISA ASSAY Kit

Ras Activation ELISA ASSAY Kit

Upstate (Millipore)

96 assays

Ras proteins function as GDP/GTP-regulated binary switches in signal transduction cascades that can lead to cell growth, proliferation, differentiation, or survival. This Ras superfamily has at least five major branches that include Ras, Rho, Ran, Arf/Sar, Rab. Ras itself has 3 primary isoforms (H-Ras, N-Ras, and K-Ras) that differ in their approximately 20 C-terminal amino acids. They are collectively referred to as Ras. In its active form, Ras is bound to GTP. This causes a conformational change that allow it to interact and bind to several effector molecules, most notably the members of the Raf family, the RalGDS family, and Phosphoinositide 3-kinases (PI3 Kinase). Ras then cleaves GTP to GDP resulting in its inactivation. In its oncogenic, mutated state, Ras is unable to hydrolyze GTP to GDP, thus staying in an active state and activating numerous pathways.
Due to its role as a key regulator of cellular functions and its implications in various cancers, Ras has been a popular target for cancer research and anti-cancer therapeutics for the past two decades for both academic and pharmaceutical research. It was also the first human oncogene identified. Mutations in cellular Ras have been found to be present in a large percentage of all human cancers. More specifically, K-Ras mutations occur frequently in lung, pancreatic, and colon cancers, where as H-Ras mutations are prevalent in bladder, kidney, and thyroid cancers, and N-Ras mutations are associated with melanoma, hepatocellular carcinoma, and leukemia (3). In recent studies, Ras activity has also been implicated in a number of human development defects such as Costello syndrome and Noonan syndrome (4).
One path that the pharmaceutical industry has taken to control Ras and its activity is by attacking what some consider its Achilles’ heel. For its activation, Ras must localize to the plasma membrane, but interestingly, it lacks a transmembrane domain. To achieve this, Ras must first undergo a post-translational modification known as prenylation or geranylation at its C-terminal CAAX motif. These drugs have yet to pass clinical trials though and there is doubt that they will ever be successful in treating tumors associated with Ras activation.

• ELISA
• Cell Function Assay

• Human
• Mouse
• Rat

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

K-Ras
This gene, a Kirsten ras oncogene homolog from the mammalian ras gene family, encodes a protein that is a member of the small GTPase superfamily. A single amino acid substitution is responsible for an activating mutation. The transforming protein that results is implicated in various malignancies, including lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas and colorectal carcinoma. Alternative splicing leads to variants encoding two isoforms that differ in the C-terminal region. [provided by RefSeq]
H-Ras
This gene belongs to the Ras oncogene family, whose members are related to the transforming genes of mammalian sarcoma retroviruses. The products encoded by these genes function in signal transduction pathways. These proteins can bind GTP and GDP, and they have intrinsic GTPase activity. This protein undergoes a continuous cycle of de- and re-palmitoylation, which regulates its rapid exchange between the plasma membrane and the Golgi apparatus. Mutations in this gene cause Costello syndrome, a disease characterized by increased growth at the prenatal stage, growth deficiency at the postnatal stage, predisposition to tumor formation, mental retardation, skin and musculoskeletal abnormalities, distinctive facial appearance and cardiovascular abnormalities. Defects in this gene are implicated in a variety of cancers, including bladder cancer, follicular thyroid cancer, and oral squamous cell carcinoma. Multiple transcript variants, which encode different isoforms, have been identified for this gene. [provided by RefSeq]

Routinely evaluated by ELISA.

• 1. Glutathione-Coated Plate, 96-well strip format: (Part No. 3007063) 1 plate containing 12 x 8 strip-wells coated with glutathione.
• 2. Anti-Ras Detection Antibody: (Part No. 2006992) One vial containing 10 μL of anti-Ras mouse monoclonal primary antibody. Can specifically detect K-, H-, N- isoforms of Ras.
• 3. Mg2+ Lysis/Wash Buffer, 5X: (Part No. CS202617) 1 vial containing 18 mL of 5X Mg2+ Lysis/Wash Buffer for preparation of cell lysate.
• 4. 20% Tween® 20: (Part No. # 20-246) One vial containing 3 mL of 20% Tween® 20.
• 5. 10% BSA in TBS (Blocking Buffer): (Part No. 20-191) One bottle containing 10 mL 10% BSA in 1X TBS.
• 6. 20X TBS Wash: (Part No. 20-190) One bottle containing 50 mL of 20X TBS, pH 7.4.
• 7. Gt x Ms, HRP: (Part No. CS202619) One amber vial containing 25 µL of goat anti-mouse HRP conjugated antibody.
• 8. Chemiluminescent Reaction Buffer: (Part No. 90496) One amber bottle containing 8 mL of Chemiluminescent Reaction Buffer. Keep out of light.
• 9. Chemiluminescent Detection Reagent: (Part No. 90495) One bottle vial containing 4 mL of Chemiluminescent Detection Reagent. Keep out of light.
• Store at -80º C
• 10. Raf-1-RBD, GST: (Part No. 14-863) 2 vials containing 150 μL of Raf-1-RBD at 2 μg/μL.
• 11. EGF stimulated HeLa Cell Lysate: (Part No. 12-500) 2 vials containing 40 µL of lysate at 5 mg/mL concentration, lysed in Mg2+ Lysis/Wash buffer (Catalog # CS202617).

Upstate

P01116

17-424

Ras proteins are small GTP-binding proteins which, unlike the heterotrimeric G-proteins, contain all GTPase and effector functions within a single polypeptide. At least three isoforms of Ras exist, Ki-Ras, Ha-Ras and N-Ras, with distinct expression patterns but similar signaling activity. Ras is palmitoylated and farnesylated at the carboxy terminus, anchoring it in the membrane. In resting cells, Ras is loaded with GDP, and is activated subsequent to growth factor stimulation of receptors, which recruit Ras Guanine nucleotide Exchange Factors to the plane of the membrane. Proximity of exchange factors to the Ras proteins causes release of GDP, and its replacement by GTP. In its GTP-bound form, Ras binds several proteins, including Raf, RalGDS and PI3 Kinase. Inactivation of Ras occurs by GTP hydrolysis, which is greatly accelerated by RasGAP or NF-1, two known Ras GTPase Activating Proteins. It is possible to assay for Ras activation by incubation of lysates with the Ras-binding domain of Raf-1, which selectively binds to Ras:GTP.

• KRAS
• K-Ras
• v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
• K-ras p21 protein
• HRAS
• H-Ras
• Ha-Ras
• NRAS
• N-Ras
• neuroblastoma RAS viral (v-ras) oncogene homolog

Ras Activation ELISA ASSAY Kit

• NP_203524.1
• NP_001123914.1
• NP_002515.1

1. Multi-channel or repeating pipettes
2. Plate shaker at room temperature and at 4oC.
3. Pipettors & tips capable of accurately measuring 1-1000 μL
4. Graphing software for plotting data or graph paper for manual plotting of data
5. Multi-channel pipettor reservoirs
6. Protease inhibitors
7. Nuclease free water/ deionized water
8. PBS
9.Luminometer or CCD camera coupled imaging system