Cdc42 G-LISA Activation Assay Kit (colorimetric)

Product Uses Include

• Cdc42 signaling pathway studies
• Cdc42 activation assays with primary cells
• Studies of Cdc42 activators and inactivators
• Cdc42 activation assays with limited material
• High throughput screens for Cdc42 activation

Introduction
The G-LISA series of Small G-Protein Activation Assays are ELISA based assays with which you can measure the GTP form of small G-proteins from lysates of cells or tissues and all in less than 3 h. The Cdc42 G-LISA Activation Assay measures the entire level of GTP-loaded Cdc42 protein in cell lysates. The level of activation is measured by reading at OD490nm. 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 please check webpage BK124, for Rac1 go to BK128 for Rac1, 2, 3 go to BK125 for RalA go to BK129.

The Cdc42 G-LISA Activation Assay is very sensitive and has excellent accuracy for duplicate samples. See this section on our G-LISA information resource page.

Example results
Serum starved Swiss 3T3 cells were stimulated with the Cdc42 activating compound EGF and Cdc42 activation was measured with the G-LISA method (Fig 1 and 2).

Figure 1. Cdc42 activation by EGF measured by the Cdc42 G-LISA Activation Assay. Swiss 3T3 cells were serum starved (SS) for 16 h at 1% serum and 8 h with 0% serum and treated with EGF (100 ng/ml for 2 min). Cell lysates (8, 17, 35 ug) were subjected to the G-LISA assay. Data was read at 490 nm. Numbers on top the yellow columns indicate the fold increase in signal caused by EGF activation, you will notice the ratio remains the same with different protein loadings indicating good linearity with different protein loadings. 500 ug of the same lysates were subjected to the traditional PAK pull-down assay (Cat.# BK034) with similar results.

Figure 2. Time course of Cdc42 activation using EGF at 10, 50 and 100ng/ml . Swiss 3T3 cells were serum starved (SS) for 16 h at 1% serum and 8 h with 0% serum and treated with EGF (10, 50 and 100 ng/ml for1.5, 3.0, 6.0, 10 and 30 min). Cell lysates subjected to the G-LISA assay and OD was read at 490 nm. The "controlled state" serum starved value (0.22) was subtracted from these samples prior to plotting. At 100 ng/ml the total activation was 2.1 fold or 110% over the controlled state at 1.5 min. This type of analysis can be performed in one afternoon.

Kit contents
The kit contains sufficient reagents to perform 96 Cdc42 activation assays. Since the Cdc42-GTP affinity wells are supplied as strips and the strips can be broken into smaller pieces, each kit can be used for anywhere from 1 to 96 assays. The following components are included in the kit:

1. 96 Cdc42-GTP affinity wells (divisible into 12 strips of 8 wells each)
2. Lysis buffer
3. Antigen presenting buffer
4. Wash buffer
5. Antibody dilution buffer
6. Anti-Cdc42 antibody (Cat.# ACD03)
7. HRP-labeled secondary antibody
8. Positive control Cdc42 protein
9. Protease inhibitor cocktail (Cat. # PIC02)
10. Color development reagents
11. Precision Red Advanced protein assay reagent (Cat. # ADV02)
12. 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 (see Biohit pipettors)
3. Two orbital microplate shakers capable of 400 rpm shaking (200 to 400 rpm is the possible range)
   Examples are:

1. 96-well plate spectrophotometer capable of reading 490 nm wavelength
2. Multichannel or multidispensing pipettor (see Biohit pipettors)
3. Two orbital microplate shakers capable of 400 rpm shaking (200 to 400 rpm is the possible range)
   Examples are:

Model # 4625 Titer Plate Shaker, Lab-Line Instruments, Barnstead Intl.(average priced)

Model # RF7854 Digital Microplate Shaker, ML Market Lab, researchml.com (economical priced)

Model # RF7855 Incubating Microplate Shaker, ML Market Lab, researchml.com (deluxe model)

Go to main G-LISA page

G-LISA Products:
Rac1, 2, 3 G-LISA Activation Assay, colorimetric format (Cat.# BK125)
Rac1 G-LISA Activation Assay, luminescence format (Cat.# BK126)
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)

G-LISA is a registered trademark of Cytoskeleton, Inc (CO). All rights reserved.