The Thermo Scientific Pierce Active Rap1 Pull-Down and Detection Kit is a complete kit for selective enrichment and detection of GTP-bound Rap1 GTPase through specific protein interaction with the RalGDS protein-binding domain.
The Active Rap1 Pull-Down and Detection Kit includes purified GST-RalGDS Rap-binding domain (RBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/wash buffer, anti-Rap1 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 Rap1 activity.
- Highly sensitive and accurate – optimized reagents, specific anti-Rap1 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
- Validated – functionally tested for Rap1 detection to ensure quality and performance
- Compatible – effective with a variety of cell types from mouse, rat and human sources
- Follow activation of Rap1 GTPase during cell differentiation, migration, division and cytoskeletal rearrangement
- Study active Rap1 signaling in cell junctions and adhesions
- Monitor Rap1 activity after stimulation with growth factors
- Screen small molecule inhibitors for their effects on Rap1 activity
The Active Rap1 Pull-Down and Detection Kit was validated for function and specificity of the active Rap1 enrichment method using cell lysates treated with GTPγS to activate endogenous Rap1 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Rap1 in the GTP-bound form (active), resulting in a strong signal when endogenous Rap1 is present. GDP treatment pushes Rap1 into the GDP-bound state (inactive), resulting in minimal or no signal, regardless of Rap1 protein levels. This kit was optimized for Western blot detection using an HRP-conjugated secondary antibody (Goat Anti-rabbit IgG, Part No. 31460) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). The kit contains sufficient components for 30 pull-down assays.
||Rap1 is induced quickly after serum re-introduction. NIH 3T3 mouse fibroblasts were serum-starved for 24 hours before adding 10% serum. Rap1 activity was monitored for 1 hour at the indicated time points using the Active Rap1 Pull-Down and Detection Kit. The Western blot results show that Rap1 activation occurs soon after serum introduction (1 minute) and diminishes within 2 minutes.
The Rap GTPases are part of the Ras family of GTPases and are encoded by Rap1a, Rap1b and Rap2. Rap GTPases are structurally similar to Ras GTPases and have similar effector and activator proteins, although Rap GTPases have different functional activities than Ras. While Ras is involved in cell proliferation and survival, Rap1 regulates cytoskeletal rearrangements, cell adhesion and cell junction formation.
The specificity of Rap1 and Ras is mediated by their respective upstream regulators and downstream effectors. The GEFs for Rap contain a CDC25 homology domain that mediates the GDP/GTP exchange reaction and a REM domain (Ras exchange motif). Some Rap GEFs include C3G, Epac1 and 2, RasGRP2, PDZ-GEF1 and 2 and PLCε. The binding domain used in this kit is RalGDS, a GEF that contains an RA domain to which Rap1 has a higher binding affinity than Ras. The RapGAPs, including Rap1GAP and the Spa-1 family, insert an asparagine side chain into the nucleotide-binding pocket to catalyze the GTP hydrolysis reaction. Rap effector proteins, including RAPL, Riam, AF-6, Krit1, RacGEFs, Tiam1, Vav2, Rho GAPs and ARAP3, are involved in cell-cell junctions and adhesion and are often localized to the membrane or at cell-cell junctions. Besides Ras, there is also cross-talk between Rap and Rho GTPases.
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- Takai, Y., et al. (2001). Small GTP-binding proteins. Physiol Rev 81:153-208.
- Zwartkruis, F.J. and Bos J.L. (1999). Ras and rap1: Two highly related small GTPases with distinct function. Exp Cell Res 253:157-65.
- Foschi, M., et al. (1997). Biphasic activation of p21ras by endothelin-1 sequentially activates the erk cascade and phosphatidylinositol 3-kinase. EMBO J 16:6439-51.
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