Cat. # BK124

Product Uses Include

• Rho signaling pathway studies
• Rho activation assays with primary cells
• Studies of Rho activators and inactivators
• Rho activation assays with limited material
• High throughput screens for Rho activation
  
  

Introduction
The G-LISA Rho activation assays are ELISA based Rho activation assays with which you can measure Rho activity in cells in less than 3 h. BK124 is very sensitive and has excellent accuracy between duplicate samples. For a more detailed introduction on G-LISA™ assays and a listing of other available G-LISA™ kits, see our main G-LISA™ page. The BK124 Rho activation assay kit measures the level of GTP-loaded RhoA only in cells. The level of activation is measured with absorbance set at 490nm. For a kit to measure RhoA activation with luminescence detection, see Cat. # BK121.
See this section on our G-LISA™ information resource page.

Examples of publications that have cited the use of Cat.# BK124

Warren JC, Rutkowski A. and L. Cassimeris. 2006. Infection with replication-deficient adenovirus induces changes in the dynamic instability of host cell microtubules. Mol. Biol. Cell, 17, p3557-3568.

Kit contents
The kit contains sufficient reagents to perform 96 RhoA activation assays. Since the Rho-GTP affinity wells are supplied as strips and the strips can be broken into smaller pieces, each kit can be used for anywhere from one to multiple assays. The following components are included in the kit:

1. 96 Rho-GTP affinity wells (divisible into 12 strips of 8 wells each for multiple uses)
2. Lysis buffer
3. Binding buffer
4. Antigen presenting buffer
5. Wash buffer
6. Antibody dilution buffer
7. Anti-RhoA antibody
8. HRP-labeled secondary antibody
9. Positive control RhoA protein
10. Protease inhibitor cocktail
11. Absorbance detection reagents
12. Precision Red™ Advanced protein assay reagent (Cat. # ADV02)
13. Manual with detailed protocols and extensive troubleshooting guide
    
    

Equipment needed

1. 96-well plate spectrophotometer capable of reading 490 nm wavelength
2. Multichannel or multidispensing pipettor
3. Orbital microplate shaker capable of at least 200 rpm shaking (400 rpm is optimal)
   
   

Example results
Serum starved Swiss 3T3 cells were stimulated with the Rho activating compound calpeptin and RhoA activation was measured with the G-LISA method (Figures 1 and 2)

Figure 1. RhoA activation by calpeptin measured by G-LISA™ kit BK124. Swiss 3T3 (mouse) cells were serum starved for 24 h and treated with calpeptin (Cal; 0.1 mg/ml for 30 min) or DMSO carrier only (SS). 10 µg of cell lysates were subjected to the G-LISA™ assay. Absorbance was read at 490 nm.

Figure 2. Rho activity measured in Swiss 3T3 cells treated with the Cell Permeable Rho Inhibitor (CT04) using the RhoA G-LISA Activation Assay (Cat.# BK124). Serum starved Swiss 3T3 fibroblasts were untreated (no CT04) or treated with 0.20, 0.50 and 2.0 µg/ml of CT04 for 4h in serum free medium at 37°C, then activated with 100µg/ml calpeptin for 10min.  Cells were then lysed and RhoA activity was measured by the RhoA G-LISA Activation Assay (Cat.# BK124).  Note: At 2.0 µg/ml CT04 for 4h results in almost complete (90%) inhibition of RhoA activity.

Go to main G-LISA™ page

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, luminescence format (Cat.# BK121)

Associated Products:
Anti-Cdc42 monoclonal antibody (Cat.# ACD03)
Anti-Rac1 monoclonal antibody (Cat.# ARC03)
Anti-RhoA monoclonal antibody (Cat.# ARH03)