The Thermo Scientific Active Cdc42 Pull-Down and Detection Kit is a complete kit for selective enrichment and detection of GTP-bound Cdc42 GTPase through specific protein interaction with the Pak1 protein-binding domain.
The Active Cdc42 Pull-Down and Detection Kit includes purified GST-Pak1 protein-binding domain (PBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/wash buffer, anti-Cdc42 primary antibody, sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH 3T3 cells, a cell line that is known to have robust Cdc42 activity.
- Highly sensitive and accurate – optimized reagents, specific anti-Cdc42 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
- Validated – functionally tested for Cdc42 detection to ensure quality and performance
- Compatible – effective with a variety of cell types from mouse, rat and human sources
- Follow activation of Cdc42 GTPase during cell differentiation, migration, division, and cytoskeletal rearrangement
- Study the activation of Cdc42 during filopodia formation
- Monitor Cdc42 activity after stimulation with growth factors
- Screen small molecule inhibitors for their effects on Cdc42 activity
The Active Cdc42 Pull-Down and Detection Kit was validated for the function and specificity of the active Cdc42 enrichment method using cell lysates treated with GTPγS to activate endogenous Cdc42 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Cdc42 in the GTP-bound, active form, resulting in a strong signal when endogenous Cdc42 is present. GDP treatment pushes Cdc42 into the GDP-bound, inactive state, resulting in minimal or no signal regardless of Cdc42 protein levels. This 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). The kit contains sufficient components for 30 pull-down assays.
|Specificity and robustness of Thermo Scientific Pierce Active Cdc42 Pull-Down and Detection Kit. NIH 3T3 cell lysate was treated with GTPγS (activator) or GDP (inactivator). Active Cdc42 was enriched by pull-down assay. Half of each eluate (25µL) and 40µg of total lysate were analyzed by Western blot using the anti-Cdc42 antibody supplied in the kit. Only active Cdc42 was detected, as indicated by the lower signal intensity in the GDP-treated sample.
Rho family GTPases serve many cellular functions, including cell signaling, transcriptional regulation and organization of the actin cytoskeleton. This family of GTPases comprise Rho (RhoA, RhoB, and RhoC), Rac (Rac1, Rac2, Rac3, and RhoG), Cdc42 (Cdc42 and G25K), Rnd (Rnd1, Rnd2, and RhoE/Rnd3), RhoBTB family and the Miro family. These GTPases enable signal transduction from the plasma membrane to the cytosol through GPCR, tyrosine kinase, cytokine and adhesion receptors. Attachment to the plasma membrane is accomplished through geranylgeranyl lipid modifications at the carboxy-terminus of the protein. Cdc42 activation results in the polymerization of actin filaments and filopodia formation.
The Cdc42 subfamily of RhoGTPase has been less well-characterized than the Rho and Rac families. Both Cdc42 and Rac1 are activated through tyrosine receptor kinase signaling leading to SAPK and p38 stress kinase pathway activation. Cdc42 is also activated by the chemoattractant fMLP in neutrophils. Fibronectin activates Cdc42 and Rac1 to induce cell spreading, and stimulation with TNF-alpha and IL-1 results in changes in the actin cytoskeleton. There is significant cross-talk between Cdc42 and Rac1, as they act in overlapping pathways, and in some cases, Cdc42 may act upstream of Rac1 during signal transduction. Some of the main effector proteins of Cdc42 are Pak1, N-WASP and IQGAP. Pak1 is a kinase involved in the activation of JNK in the SAPK stress pathway. N-WASP is an effector that induces filopodia formation, and IQGAP interacts with F-actin filaments. In differentiating neurons, Cdc42 plays an active role in neurite outgrowth. However, the Rho family of GTPases can work agonistically during cell signaling and antagonistically during differentiation.
- Hall, A. and Lalli, G. (2010). Rho and Ras GTPases in axon growth, guidance, and branching. Cold Spring Harb Perspect Biol 2:a001818.
- Williams, D.A., et al. (2008). Rho GTPases and regulation of hematopoietic stem cell localization. Methods Enzymol 439:365-93.
- Govek, E.E., et al. (2005). The role of the Rho GTPases in neuronal development. Genes Dev 19:1-49.
- Wennerberg, K. and Der, C.J. (2004). Rho-family GTPases: It's not only Rac and Rho (and I like it). J Cell Sci 117:1301-12.
- Benard, V. and Bokoch, G.M. (2002). Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol 345:349-59.
- Bar-Sagi, D. and Hall, A. (2000). Ras and Rho GTPases: A family reunion. Cell 103:227-38.
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