- ¥5500
- cytoskeleton
- USA
- BK035-S
- 2025年12月03日
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- 详细信息
- 技术资料
- 英文名：
Rac1 Pulldown Activation Assay Kit
- 供应商：
研卉生物
- 规格：
20 assays
Product Uses Include
- Analysis of in vivo Rac1 activation levels.
- Detection of compounds and proteins that enhance Rac1 activity.
- Detection of compounds and proteins that inhibit Rac1 activity
Introduction
The Rho switch operates by alternating between an active, GTP-bound state and an inactive, GDP-bound state. Understanding the mechanisms that regulate activation / inactivation of the GTPases is of obvious biological significance and is a subject of intense investigation. The fact that many Rho family effector proteins will specifically recognize the GTP bound form of the protein has been exploited experimentally to develop a powerful affinity purification assay that monitors Rac and Cdc42 protein activation. The assay uses the Cdc42/Rac Interactive Binding (CRIB) region (also called the p21 Binding Domain, PBD) of the Cdc42 / Rac effector protein, p21 activated kinase 1 (PAK). The CRIB/PBD protein motif has been shown to bind specifically to the GTP-bound form of Rac and/or Cdc42 proteins. The fact that the PBD region of PAK has a high affinity for both GTP-Rac and GTP-Cdc42 and that PAK binding results in a significantly reduced intrinsic and catalytic rate of hydrolysis of both Rac and Cdc42 make it an ideal tool for affinity purification of GTP-Rac and GTP-Cdc42 from cell lysates. The PAK-PBD protein supplied in this kit corresponds to residues 67-150. This includes the highly conserved CRIB region (aa 74-88) plus sequences required for the high affinity interaction with GTP-Rac and GTP-Cdc42. The PAK-PBD is in the form of a GST fusion protein, which allows one to "pull-down" the PAK-PBD/GTP-Rac (or GTP-Cdc42) complex with glutathione affinity beads. The assay therefore provides a simple means of quantitating Rac or Cdc42 activation in cells. The amount of activated Rac is determined by a Western blot using a Rac-specific antibody.
Kit contents
The kit contains sufficient materials for 20 assays, depending on assay setup, and includes reagents for positive and negative controls. A larger 50 assay version of this kit is available as Cat. # BK035-S. The following components are included:
- GST-tagged PAK-PBD protein on colored agarose beads (Cat. # PAK02)
- Rac1 monoclonal antibody (Cat. # ARC03)
- His-tagged Rac1 protein (Cat. # RC01)
- GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
- GDP
- Cell lysis Buffer
- Wash Buffer
- Loading Buffer
- STOP Buffer
- Protease inhibitor cocktail (Cat. # PIC02)
- Manual with detailed protocols and extensive troubleshooting guide
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Figure 1. The brightly colored glutathione agarose beads in BK035-S makes the kit easy to use. |
Equipment needed
- SDS-PAGE minigel system and western blotting transfer apparatus
Example results
The Rac1 activation assay was tested by loading the Rac1 protein in cell lysates with either GTPγS or GDP. As expected, the GTPγS-loaded Rac1 is very efficiently precipitated while very little GDP-loaded Rac1 is precipitated (Fig. 2).
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Figure 2. Results from BK035-S Rac1 activation assay. Activated Rac1 was precipitated and detected in a Western blot using kit BK035-S. The first lane shows a 50 ng recombinant His-tagged Rac1 standard (Recombinant His-Rac1). The following lanes shows the pull-down of inactive, GDP-loaded Rac1 (Rac1-GDP PD) or active, GTPγS-loaded Rac1 (Rac1-GTP PD) from equal amounts of cell lysates. |
Please check out the new version of the Rac Activation Assay and associated products:
G-LISA™ Products:
Cdc42 G-LISA™ Activation Assay, colorimetric format (Cat.# BK127)
Rac1 G-LISA™ Activation Assay, luminescence format (Cat.# BK126)
Rac1,2,3 G-LISA™ Activation Assay, colorimetric format (Cat.# BK125)
RhoA G-LISA™ Activation Assay, colorimetric format (Cat.# BK124)
RhoA G-LISA™ Activation Assay, luminescence format (Cat.# BK121)
Associated Products:
Anti-Cdc42 monoclonal antibody (Cat.# ACD03)
Anti-Rac1 monoclonal antibody (Cat.# ARC03)
Anti-RhoA monoclonal antibody (Cat.# ARH03)
Lee et al., 2012. Barrier protective effects of withaferin A in HMGB1-induced inflammatory responses in both cellular and animal models. Toxicol. Appl. Pharmacol. v 262, pp 91-98.
Nithipatikom et al., 2012. Cannabinoid Receptor Type 1 (CB1) Activation Inhibits Small GTPase RhoA Activity and Regulates Motility of Prostate Carcinoma Cells. Endocrinology. v 153, pp 29-41.
Wong et al., 2012. Merlin/NF2 Regulates Angiogenesis in Schwannomas through a Rac1/Semaphorin 3F-Dependent Mechanism. Neoplasia. v 14, pp 84–94.
Jayaram et al., 2011. Isoprenylcysteine carboxyl methyltransferase facilitates glucose-induced Rac1 activation, ROS generation and insulin secretion in INS 832/13 β-cells. Islets. v 3, pp 48-57.
Skalski et al., 2011. SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover. Biochimica et Biophysica Acta - Mol. Cell Res. v 1813, pp 148-158.
Ock et al., 2011. A novel approach for stress-induced gastritis based on paradoxical anti-oxidative and anti-inflammatory action of exogenous 8-hydroxydeoxyguanosine. Biochem. Pharmacol. v 81, pp 111-122.
Slice et al., 2005. Angiotensin II and epidermal growth factor induce cyclooxygenase-2 expression in intestinal epithelial cells through small GTPases using distinct signaling pathways. J. Biol. Chem. v 280, pp 1582-1593.
Sasai et al., 2004. The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements. Nat. Cell Biol. v 6, pp 741-748.
Yang et al., 2004. Rho and Rho-kinase mediate thrombin-induced phosphatidylinositol 4-phosphate 5-kinase trafficking in platelets. J. Biol. Chem. v 279, pp 42331-42336.
Zhang et al., 2004. Distinct roles of two structurally closely related focal adhesion proteins, α-parvins and β-parvins, in regulation of cell morphology and survival. J. Biol. Chem. v 279, pp 41695-41705.
Chromy et al., 2003. Self-assembly of Aβ(1-42) into globular neurotoxins. Biochemistry. v 42, pp 12749-12760.
Quadri et al., 2003. Endothelial barrier strengthening by activation of focal adhesion kinase. J. Biol. Chem. v 278, pp 13342-13349.
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