Biochem Kits™
Rac1,2,3 G-LISA™ Activation Assay (colorimetric)

Cat. # BK125

Product Uses Include

  • Rac signaling pathway studies
  • Rac activation assays with primary cells
  • Studies of Rac activators and inactivators
  • Rac activation assays with limited material
  • High throughput screens for Rac 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. 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 Rac1,2,3 G-LISA™ Activation Assay measures the entire level of GTP-loaded Rac1,2 and 3 protein in cell lysates, this is in contrast to Cat.# BK126 which measures only Rac1 activation levels. The level of activation is measured with absorbance at 490nm. For a kit to measure RhoA activation please check webpage BK124.

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


Examples of publications that have cited the use of Cat.# BK125 are described below:

Ramirez SH, Heilman D, Morsey B, Potula R, Haorah J and Persidsky Y. Activation of Peroxisome Proliferator-Activated Receptor (PPAR) Suppresses Rho GTPases in Human Brain Microvascular Endothelial Cells and Inhibits Adhesion and Transendothelial Migration of HIV-1 Infected Monocytes. J. Immunology, 2008, 180, p1854-1865.

Pontow S, Harmon B, Campbell N, and Ratner L. Antiviral Activity of a Rac GEF inhibitor Characterized with a Sensitive HIV/SIV Fusion Assay. Virology. 2007 November 10; 368(1): 1–6.

Zhou X, Tian F, Sandze J, Cao R, Flaberg E, Szekely L, Cao Y, Ohlsson C, Bergo M, Bore´ n J and Akyu¨ rek  LM.    Filamin B deficiency in mice results in skeletal malformations and impaired microvascular development.  PNAS, March 6th, 2007, 104, (10), p3919–3924.

Schlegel N, Burger S, Golenhofen N, Walter U, Drenckhahn D, and Waschke J. The role of VASP in the regulation of cAMP- and Rac 1-mediated endothelial barrier stabilization. Am J Physiol Cell Physiol (November 7, 2007). doi:10.1152/ajpcell.00273.2007


Kit contents
The kit contains sufficient reagents to perform 96 Rac activation assays. Since the Rac-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 Rac-GTP affinity wells (divisible into 12 strips of 8 wells each)
  2. Lysis buffer
  3. Binding buffer
  4. Antigen presenting buffer
  5. Wash buffer
  6. Antibody dilution buffer
  7. Anti-Rac antibody
  8. HRP-labeled secondary antibody
  9. Positive control Rac 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 Rac activating compound EGF and Rac activation was measured with the G-LISA method (Fig 1 and 2).

Figure 1. Rac activation by EGF measured by G-LISA™ kit BK125. Swiss 3T3 (mouse) cells were serum starved for 24 h and treated with EGF (Cal; 10ng/ml for 3 min) or buffer only (SS). 10 µg of cell lysates were subjected to the G-LISA™ assay. Absorbance was read at 490 nm.


Figure 2. Rac  activation by EGF measured by G-LISA™.  Swiss 3T3 cells were serum starved (SS) for 24 h and treated with EGF (10 ng/ml for 2 min). 20, 10, 5, 2.5, 1.25 µg of cell lysates were subjected to the G-LISA™ assay. Absorbance was read at 490 nm. 500 µg of the same lysates were subjected to the traditional PAK pull-down assay (shown in inset, Cat.# BK035).


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)
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)
Product description Cat. # Amount Price & Order
Rac1,2,3 G-LISA™ Activation Assay, colorimetric. BK125 96 assays