The Thermo Scientific Active Rac1 Pull-Down and Detection Kit is a complete kit for the selective enrichment and detection of GTP-bound Rac1 GTPase through specific protein interaction with the Pak1 protein-binding domain.
The Active Rac1 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-Rac1 primary antibody, SDS sample buffer, spin columns and collection tubes. The kit was validated using lysates from NIH3T3 cells, a cell line that is known to have robust Rac1 activity.
- Highly sensitive and accurate – optimized reagents, specific anti-Rac1 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
- Validated – functionally tested for Rac1 detection to ensure quality and performance
- Compatible – effective with a variety of cell types from mouse, rat and human sources
- Follow activation of Rac1 GTPase during cell differentiation, migration, division and cytoskeletal rearrangement
- Study Rac1 dependent lamellipodia formation
- Study the role of active Rac1 in cancer and angiogenesis
- Monitor Rac1 activity after stimulation with growth factors
- Screen small molecule inhibitors for their effect on Rac1 activity
The Active Rac1 Pull-Down and Detection Kit was validated for function and specificity of the active Rac1 enrichment method using cell lysates treated with GTPγS to activate endogenous Rac1 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Rac1 in the GTP-bound, active form, resulting in a strong signal when endogenous Rac1 is present. GDP treatment pushes Rac1 into the GDP-bound, inactive state, resulting in minimal or no signal, regardless of Rac1 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). The kit contains sufficient components for 30 pull-down assays.
|Rac1 promotes the early formation of neurites. NS-1 neuronal cells (PC-12 derivative) were differentiated with 50ng/mL neuronal growth factor (NGF) and analyzed for Rac1 activation (A) and neurite formation (C). The robustness of the Active Rac1 Pull-Down and Detection Kit is demonstrated through the monitoring of endogenous Rac1 activity in differentiating cells. The fluorescent images show the benefit of using the Pak1-binding domain to detect localization of active Rac1. Panel A: Activated Rac1 was enriched by the pull-down assay and detected by Western blot. Panel B: Densitometry of the active Rac1 Western blot. Data was normalized to total Rac1 for each time point. Panel C: Localization of Rac1 and Pak1-PBD effectors during cell differentiation. Cells were fixed at 2 days post-stimulation, permeabilized and stained for immunofluorescence. Total Rac1 was detected using the anti-Rac1 antibody included in the kit and Thermo Scientific DyLight 549-conjugated goat anti-mouse IgG. Domain interactors of Pak1 were stained using GST-tagged Pak1 PBD and DyLight 488-conjugated anti-GST antibody. Co-localization of Rac1 with Pak1-PBD in the merged image suggests that active Rac1 localizes to neurites.
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. Rac1 activation results in actin polymerization and appears as membrane ruffling at the cellular periphery. Rac1 activation also results in lamelipodia formation.
The Rac1 GTPase transduces signals through tyrosine kinases, adhesion molecules or cytokine/chemokine receptors after stimulation with growth factors (EGF, insulin, PDGF, NGF), integrins (fibronectin) or chemoattractants (fMLP). For example, stimulation with EGF results in PI3 kinase activation, resulting in cell growth and reorganization at the cell periphery (membrane ruffling). Alternatively, tyrosine receptor kinase signaling through Rac1 leads to activation of the MAPK stress response pathways SAPK (JNK) and p38. Stimulation of cells with fibronectin results in integrin-mediated cell spreading. Two of the main effector proteins of Rac1 are Pak1 and phosphoinositol 4-phosphate 5-kinase. Pak1 (p65 Pak) is a kinase that activates the JNK pathway, while phosphoinositol 4-phosphate 5-kinase promotes actin filament assembly. Rac is critical for T-cell development and for promoting differentiating cells. However, the Rho family of GTPases can work agonistically during cell signaling and 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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