Biochem Kits™
Rac1 G-LISA™ Activation Assay (luminescence)

Cat. # BK126

Product Uses Include

  • Rac1 signaling pathway studies
  • Rac1 activation assays with primary cells
  • Studies of Rac1 activators and inactivators
  • Rac1 activation assays with limited material
  • High throughput screens for Rac1 activation/inhibition

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 level of activation is measured with luminescence. 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 G-LISA™ Activation Assay (Cat.# BK126) measures the level of GTP-loaded Rac1 protein in cell lysates, this is in contrast to Cat. BK125 which does not distinguish between activated Rac1,2 or 3. For a kit to measure RhoA activation please check webpage BK124.

The Rac1 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 (similar to BK126)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 96 individual wells)
  2. Lysis buffer
  3. Binding buffer
  4. Antigen presenting buffer
  5. Wash buffer
  6. Antibody dilution buffer
  7. Anti-Rac1 antibody
  8. HRP-labeled secondary antibody
  9. Positive control Rac1 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. 96-well plate luminometer
  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. Rac1 activation by EGF measured by G-LISA™ kit BK126. Swiss 3T3 (mouse) cells were serum starved for 24 h and treated with EGF (10ng/ml for 2.5 min) or buffer only (SS). 10 µg of cell lysates were subjected to the G-LISA™ assay. Luminescence measured over 100 milli-second.


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). 60, 30, 15, 7.5, 2.5 µg of cell lysates were subjected to the G-LISA™ assay. Luminescence was measured over 10 milli-seconds. 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)
Rac1,2,3 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)

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 G-LISA™ Activation Assay, luminescence. BK126 96 assays