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Cytoskeleton社 RhoA / Rac1 / Cdc42の活性アッセイキット

                                   

Rhoファミリータンパク質関連の研究に 

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RhoA / Rac1 / Cdc42 Activation Assay Combo Bioc hem Kit (bead pull-down format) - 3 x 10 assays

 

Product Overview

 

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.

                                                                           

       Figure 1. Schematic of RhoA/Rac1/Cdc42 Activation (Pull-down) Assay

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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:

  1. GST-tagged Rhotekin-RBD protein on colored agarose beads (Part # RT02-S)
  2. GST-tagged PAK-PBD protein on colored agarose beads (Part # PAK02-S)
  3. RhoA-specific monoclonal antibody, 50 ug (Cat. # ARH03)
  4. Rac1-specific monoclonal antibody, 50 ug (Cat. # ARC03)
  5. Cdc42 monoclonal antibody, 50 ug (Cat. # ACD03)
  6. His-tagged RhoA protein (Part # RHWT)
  7. His-tagged Rac1 protein (Part # RCWT)
  8. His-tagged Cdc42 protein (Part # CDWT)
  9. GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
  10. GDP (Part # GDP01)
  11. Cell lysis Buffer (Part # CLB01)
  12. Wash Buffer (Part # WB01-S)
  13. Loading Buffer (Part # LB01)
  14. STOP Buffer (Part # STP01)
  15. DMSO (Part # DMSO)
  16. Protease inhibitor cocktail (Cat. # PIC02)
  17. Manual with detailed protocols and extensive troubleshooting guide

 

 

Equipment needed

  1. SDS-PAGE minigel system and Western blotting transfer apparatus

 

References

  1. Aspenstrom, P.  1999.  Effectors for the Rho GTPases.  Curr. Opin. In Cell Biol. 11, 95-102.
  2. 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.
  3. 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       

          

Product Description MSDS Datasheet Cat #
RhoA / Rac1 / Cdc42 Activation Assay Combo Biochem Kit (bead pull-down format) - 3 x 10 assays BK030
Cdc42 Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK034-S
Rac1 Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK035-S
Ras Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK008-S
RhoA Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK036-S

 

Please check out the new versions of Activation Assays and associated products:

G-LISA® Activation Assays:
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)

Large Pull-down Activation Assays:
Cdc42 Activation Assay Biochem Kit, bead pull-down format (Cat.# BK034)
Rac1 Activation Assay Biochem Kit, bead pull-down format (Cat.# BK035)
RhoA Activation Assay Biochem Kit, bead pull-down format (Cat.# 
BK036)


Associated Products:
Anti-Cdc42 monoclonal antibody (Cat.# ACD03)
Anti-Rac1 monoclonal antibody (Cat.# ARC03)
Anti-RhoA monoclonal antibody (Cat.# ARH03)
GST-tagged Rhotekin-RBD protein on colored agarose beads (Cat. # 
RT02)
GST-tagged PAK-PBD protein on colored agarose beads (Cat. # 
PAK02)
His-tagged RhoA protein (Cat. # 
RH01)
His-tagged Rac1 protein (Cat. # 
RC01)
His-tagged Cdc42 protein (Cat. # 
CD01)

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