RhoA / Rac1 / Cdc42 Activation Assay Combo Biochem Kit (bead pull-down format) - 3 x 10 assays
Manufacturer: Cytoskeleton Inc
Product Overview :
- The combo kit is an ideal choice when your experimental observations suggest that one or more Rho family proteins are activated.
- Provides 10 assays each for detection of activated RhoA, Rac1, and Cdc42.
- Includes an extensive array of reagents (see kit contents below).
- Most cost effective way to obtain a Rho family activation profile.
Assay Principle :
The principle of the assay is shown schematically below (Figure 1). The assay is based upon the fact that a Rho family effector protein is known to bind preferentially to the active (GTP-bound) form of its target GTPase (1). In the case of RhoA activation, the rhotekin-RBD effector domain is used to make the affinity beads (2). In the case of Cdc42 and Rac1 activation, the PAK-PBD effector domain is used for affinity beads (3). Example Western blot results and detailed protocols can be viewed by clicking the documents tab above and downloading the pdf datasheet.
Kit contents :
The kit contains sufficient materials for 10 assays each for RhoA, Rac1, and Cdc42 (depending on activation levels in cells), including reagents for positive and negative controls. The following components are included:
- GST-tagged Rhotekin-RBD protein on colored agarose beads (Part # RT02-S)
- GST-tagged PAK-PBD protein on colored agarose beads (Part # PAK02-S)
- RhoA-specific monoclonal antibody, 50 ug (Cat. # ARH03)
- Rac1-specific monoclonal antibody, 50 ug (Cat. # ARC03)
- Cdc42 monoclonal antibody, 50 ug (Cat. # ACD03)
- His-tagged RhoA protein (Part # RHWT)
- His-tagged Rac1 protein (Part # RCWT)
- His-tagged Cdc42 protein (Part # CDWT)
- GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
- GDP (Part # GDP01)
- Cell lysis Buffer (Part # CLB01)
- Wash Buffer (Part # WB01-S)
- Loading Buffer (Part # LB01)
- STOP Buffer (Part # STP01)
- DMSO (Part # DMSO)
- Protease inhibitor cocktail (Cat. # PIC02)
- Manual with detailed protocols and extensive troubleshooting guide
Equipment needed :
- SDS-PAGE minigel system and Western blotting transfer apparatus
- Aspenstrom, P. 1999. Effectors for the Rho GTPases. Curr. Opin. In Cell Biol. 11, 95-102.
- Ren, X.D. et al. 1999. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18, 578-585.
- Benard, V. et al. 1999. Characterization of Rac and Cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J. Biol. Chem. 274: 13198-13204 .
All prices shown are exlusive of VAT.