Rac & Cdc42 Activator

Introduction

Rac and Cdc42 Activator (Cat nr CN02) is useful for efficient activation of Rac1, Rac2, Rac3 and Cdc42 in a variety of cultured cells.  The reagent activates Rac and Cdc42 proteins in fibroblasts, neurons, epithelial, endothelial, and hematopoietic cells as well as other primary and immortalized lines.  Cells treated with the activator can be subjected to any one of a number of assays that indicate an increase in Rac or Cdc42 activity, including membrane ruffles and lamellipodia (Rac) staining (Cat nr BK005 and Figure 2 in the gallery) and Rac or Cdc42 activity assays by G-LISA™ (Cat nr BK125 and BK127)  See Figure 1 in the gallery for example of Cdc42 activation measured by the G-LISA assay.

There are many activators of Rac and Cdc42 proteins in mammalian cells. Commonly used ones are lysophosphatidylcholine (Rac), bradykinin (Cdc42), epidermal growth factor (EGF, Rac and Cdc42) and tumor necrosis factor (TNF, Cdc42). Through years of experience in Rac and Cdc42 Activation Assays, Cytoskeleton Inc. has identified EGF as a compound that activates many cell types and has a robust signal compared to basal levels. EGF is standardized in CN02 by measurement in units, thus 100 ng of EGF is 1 unit of CN02.

Material

Rac and Cdc42 Activator is greater than 95% pure which is supplied as a white lyophilized powder.

Storage

The lyophilized protein can be stored at 4°C or -70°C with less than 10% humidity for 6 months.

Biological Activity

At 0.25 to 0.5 unit / ml CN02 will activate Rac by 1.5 to 4 fold in epithelial, endothelial, hematopoietic and primary human cell types as measured by the G-LISATM Rac Activation Assay (Cat nr BK125) and observed by ruffles and lamellipodia formation (see Figure 2B in the gallery). At 0.5 to 1.0 unit / ml CN02 will activate Cdc42 by 1.25 to 2.5 fold in epithelial, endothelial, hematopoietic and primary human cell types as measured by the G-LISATM Cdc42 Activation Assay (Cat nr BK127, see Figure 1 in the gallery).

The concentration of CN02 activator required for efficient activation of Rac and Cdc42 proteins can vary between cell types and whether the medium contains serum or not.  In addition, the length of treatment can be manipulated to yield a moderate or robust activation (see Tables 1 and 2 in the gallery).  For these reasons, the concentration of this reagent and the duration of treatment should be determined by the user.  Typically the effective range is between 0.1 units / ml and 1.0 unit / ml for incubation in serum free medium. In media containing serum it might be difficult to observe the difference between CN02 treated versus untreated samples because there are activators in the serum added to cultured cells.  Inconjunction, incubation times of 1 to 10 min should be tested for each cell type.  Recommended conditions for several cell types are detailed in Tables 1 and 2 in the gallery.

Product Uses Include

• Positive control for Rac and Cdc42 activation studies.
• Study the effects of Rac and Cdc42 activation on cell motility.
• Study the effects of Rac activation on the rearrangement of the actin cytoskeleton.
• Investigate the effects of Rac and Cdc42 activation with respect to cross talk to other signal transduction pathways.