The Thermo Scientific Active Ras Pull-Down and Detection Kit enables selective enrichment and detection of GTP-bound Ras GTPase through a specific protein interaction with the Raf1 protein-binding domain.
The Active Ras Pull-Down and Detection Kit includes purified GST-Raf1 Ras-binding domain (RBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/washing buffer, anti-Ras antibody, SDS sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH 3T3 cells, a cell line known to have robust Ras activity.
- Highly sensitive and accurate – optimized reagents, specific anti-Ras antibody and Western blot procedure ensure accurate controls and semi-quantitative results
- Validated – functionally tested for Ras detection to ensure quality and performance
- Compatible – effective for a variety of cell types from mouse, rat and human sources
- Follow activation of Ras GTPase during cell differentiation, migration, division, and cytoskeletal rearrangement
- Study the role of active Ras in cancer and angiogenesis
- Monitor Ras activity after stimulation with growth factors
- Screen small molecule inhibitors for their effect on Ras activity
The Active Ras Pull-Down and Detection Kit was validated for the function and specificity of the active Ras enrichment method using cell lysates treated with GTPγS to activate endogenous Ras and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment trap Ras in the GTP-bound, active form, resulting in a strong signal when endogenous Ras is present. GDP treatment pushes Ras into the GDP-bound, inactive state, resulting in little to no signal, regardless of Ras protein levels. The kit is optimized for Western blot detection with an HRP-conjugated secondary antibody (Goat anti-Mouse IgG, Part No. 31430) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). Each kit contains sufficient components for 30 pull-down assays.
|Ras response to PDGF stimulation. NIH 3T3 cells were serum-starved for 24 hours, stimulated with platelet-derived growth factor (PDGF), and monitored for Ras GTPase activity. Panel A: The top Western blot shows the level of active GTPase isolated by pull-down assay. The lower blot shows the total amount of expressed GTPase in the lysate. The data reveal the biphasic activation of Ras Panel B: Densitometry of the active Ras Western blot. Values for active Ras are normalized to the total loading control. Read more...
Kit components can also be used for immunofluorescent staining. Neuronal NS-1 cells were stimulated with NGF to study the spatial distribution of active Ras using the GST-RBD protein and anti-Ras antibody supplied in the kit.
|Localization of Ras and Raf1 binding effectors during neuronal differentiation. NS-1 neuronal cells (PC-12 derivative) were differentiated with 50ng/mL neuronal growth factor (NGF) and analyzed for Ras activation and neurite formation. Cells were fixed at 2 days post-stimulation, permeabilized, and stained for immunofluorescence. Total Ras was detected using anti-Ras antibody and DyLight 549-conjugated goat anti-mouse IgG. Domain interactors of Raf1 were detected using GST-Raf1-RBD and DyLight 488-conjugated anti-GST IgG. Co-localization of Ras with the Raf1 PBD in the merged image shows that active Ras localizes to both neurite extensions and the perinuclear region. Read more...
The Ras superfamily of GTPases, named after the rat sarcoma viral oncogene, contains many Ras isoforms, including K-Ras, H-Ras, and N-Ras. While the three isoforms are expressed at different levels in different types of cells, in general, activating mutations of at least one of these isoforms are present in ~15% of all cancers. The Ras proteins serve as initiators of intracellular signal transduction from extracellular molecules and associate with the plasma membrane via lipid modification and prenylation of its carboxy terminus. These modifications at the carboxy terminus determine the localization of Ras to distinct membrane microdomains, which dictates subsequent downstream signaling. Ras signaling pathways affect many cellular processes including proliferation, survival, vesicular trafficking and gene expression.
Signaling through Ras is central to many cellular responses, and activation of Ras is regulated by several GEF and GAP proteins. The GEF proteins mediate GTPase activation by dissociating GDP from the inactive Ras to allow binding of Ras to GTP. Conversely, GAP proteins inactivate GTPases by hydrolyzing GTP to GDP. Ras mediates downstream signaling by interacting with effector proteins, including Raf and PI3 kinase. Raf1 is a serine/threonine protein kinase that is part of the MAP kinase kinase signaling pathway that leads to the activation of ERK and p38, which influences proliferation and survival. PI3 kinase signaling results in activation of AKT and mTOR, which are central for cell growth and survival. Ras is also integral for cellular differentiation and development, including immune cell development and function.
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- Taylor S.J. et al. (2001). Nonradioactive determination of Ras-GTP levels using activated Ras interaction assay. Methods Enzymol 333:333-42.
- Takai Y. et al. (2001). Small GTP-binding proteins. Physiol Rev 81:153-208.
- Bar-Sagi D. and Hall A. (2000). Ras and Rho GTPases: A family reunion. Cell 103:227-38.
Review of Active GTPase Pull-Down and Detection Kits
Review of Pull-down Assays
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