Cat. # BK121

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
This G-LISA™ Rho activation assay measures the combined levels of GTP-loaded RhoA, RhoB and RhoC in cells. The level of activation is measured with luminometry. The G-LISA Rho activation assays are ELISA based Rho activation assays with wich you can measure Rho activity in cells in less than 3 h. For a more detailed introduction on G-LISA™ assays and a listing of other available G-LISA™ kits, see our main G-LISA™ page. For a kit to measure RhoA activation with colorimetric detection, see Cat. # BK124.

Example publications that have cited the use of Cat.# BK121

Higashibata A, Imamizo-Sato M, Seki M, Yamazaki T. and Ishioka N. 2006. Influence of simulated microgravity on the activation of small GTPase Rho involved in cytoskeletal formation – molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factor. BMC Biochemistry, 7, (19), p1-9.

Woods A. and Beier F. 2006. RhoA/ROCK signaling regulates chondrogenesis in a context-dependent manner. J. Biol. Chem. 281, (19), p.13134-13140.

Kit contents
The kit contains sufficient reagents to perform 96 Rho 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
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. Luminescence detection reagents
12. Precision Red™ Advanced protein assay reagent (Cat. # ADV02)
13. Manual with detailed protocols and extensive troubleshooting guide
    
    

Equipment needed

1. Luminometer capable of reading a 96-well plate
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, HeLa and A431 cells were stimulated with the Rho activating compound lysophosphatidic acid and RhoA activation was measured with BK121 (Fig 1)

Figure 1. Rho activation by lysophosphatidic acid (LPA) measured by G-LISA™ kit BK121. Swiss 3T3 (mouse), A431 (human) and HeLa (human) cells were serum starved followed by stimulation by LPA.  25 µg of lysates were subjected to the G-LISA™ assay.  Data shown are relative luminescence units (RLU) over background signal (wells incubated with lysis buffer alone instead of cell lysates).  Numbers above LPA bars correspond to fold activation compared to the control serum starved samples.

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, colorimetric format (Cat.# BK124)

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