Ras Activation Assay Kit Protocol

Ras ACTIVATION ASSAY BIOCHEM KIT amount of activated Ras is determined by a quantitative western blot using a Ras Pan Ras Activation Assay Protocol, Continued.

Active Ras Detection Kit #8821

Ras Activation Assay Kit Active Ras Pull-Down Assay Perform SDS-PAGE following your preferred protocol.

E4orf1: a novel ligand that improves glucose disposal in cell culture.

Emily J Dhurandhar,Olga Dubuisson,Nazar Mashtalir,Rashmi Krishnapuram,Vijay Hegde,Nikhil V Dhurandhar

Reducing dietary fat intake and excess adiposity, the cornerstones of behavioral treatment of insulin resistance IR, are marginally successful over the long term. Ad36, a human adenovirus, offers a template to improve IR, independent of dietary fat intake or adiposity. Ad36 increases cellular glucose uptake via a Ras-mediated activation of phosphatidyl inositol 3-kinase PI3K, and improves hyperglycemia in mice, despite a high-fat diet and without reducing adiposity. Ex-vivo studies suggest that Ad36 improves hyperglycemia in mice by increasing glucose uptake by adipose tissue and skeletal muscle, and by reducing hepatic glucose output. It is impractical to use Ad36 for therapeutic action. Instead, we investigated if the E4orf1 protein of Ad36, mediates its anti-hyperglycemic action. Such a candidate protein may offer an attractive template for therapeutic development. Experiment-1 determined that Ad36 requires E4orf1 protein to up-regulate cellular glucose uptake. Ad36 significantly increased glucose uptake in 3T3-L1 preadipocytes, which was abrogated by knocking down E4orf1 with siRNA. Experiment-2 identified E4orf1 as sufficient to up-regulate glucose uptake. 3T3-L1 cells that inducibly express E4orf1, increased glucose uptake in an induction-dependent manner, compared to null vector control cells. E4orf1 up-regulated PI3K pathway and increased abundance of Ras--the obligatory molecule in Ad36-induced glucose uptake. Experiment-3: Signaling studies of cells transiently transfected with E4orf1 or a null vector, revealed that E4orf1 may activate Ras/PI3K pathway by binding to Drosophila discs-large Dlg1 protein. E4orf1 activated total Ras and, particularly the H-Ras isoform. By mutating the PDZ domain binding motif PBM of E4orf1, Experiment-4 showed that E4orf1 requires its PBM to increase Ras activation or glucose uptake. Experiment-5: In-vitro, a transient transfection by E4orf1 significantly increased glucose uptake in preadipocytes, adipocytes, or myoblasts, and reduced glucose output by hepatocytes. Thus, the highly attractive anti-hyperglycemic effect of Ad36 is mirrored by E4orf1 protein, which may offer a novel ligand to develop anti-hyperglycemic drugs.

17-218 Ras Activation Assay Kit. Download; Zoom Less. SDS; CoA; References; Brochures; Key Applications: Enzyme Assay. 17-218 1 kit Kit capacity: 30 assays.

The Ras superfamily of small GTP-binding proteins G proteins comprise a large class of proteins over 150 members that can be classified into at least five families based on their sequence and functional similarities: Ras, Rho, Rab, Arf, and Ran 1-3. These small G proteins have both GDP/GTP-binding and GTPase activities and function as binary switches in diverse cellular and developmental events that include cell cycle progression, cell survival, actin cytoskeletal organization, cell polarity and movement, and vesicular and nuclear transport 1. An upstream signal stimulates the dissociation of GDP from the GDP-bound form inactive, which leads to the binding of GTP and formation of the GTP-bound form active. The activated G protein then goes through a conformational change in its downstream effector-binding region, leading to the binding and regulation of downstream effectors. This activation can be switched off by the intrinsic GTPase activity, which hydrolyzes GTP to GDP and releases the downstream effectors. These intrinsic guanine nucleotide exchange and GTP hydrolysis activities of Ras superfamily proteins are also regulated by guanine nucleotide exchange factors GEFs that promote formation of the active GTP-bound form and GTPase activating proteins GAPs that return the GTPase to its GDP-bound inactive form 4.

The 21 kDa guanine-nucleotide binding proteins K-Ras, H-Ras, and N-Ras cycle between active GTP-bound and inactive GDP-bound forms 5. Receptor tyrosine kinases and G-protein-coupled receptors activate Ras, which then stimulates the Raf-MEK-MAPK pathway 6-8. GAP proteins normally facilitate the inactivation of Ras. However, in 30 of human tumors, point mutations in Ras prevent the GAP-mediated inhibition of this pathway 9. The most common oncogenic Ras mutation found in tumors is Gly12 to Asp G12D, which prevents Ras inactivation, possibly by increasing the overall rigidity of the protein 9,10.

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Cell Biolabs K-Ras Activation Assay Kit utilizes Raf1 RBD Agarose beads to selectively assay is critical to success. Assay Protocol. K-Ras Pull-Down Assay.

17-218 | Ras Activation Assay Kit

Ras Activation Kit ab128504 Please see Protocol: We recently purchased the abcam Ras Activation Assay kit ab128504.