Rac and Cdc42 Activator

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

Question 1: What is the chemical nature of the Rac/Cdc42 activator CN02?

Answer 1: CN02 is epidermal growth factor (EGF). The EGF is greater than 95% pure and supplied as a white lyophilized powder. One unit of CN02 is equivalent to 100 ng of EGF.

Question 2: Does CN02 selectively activate Rac or Cdc42?

Answer 2: CN02 (epidermal growth factor) will activate both Rac and Cdc42 indirectly in many different types of cells. In addition, EGF is involved in activating many other signal transduction cascades including MAPK, Akt and JNK. For a direct Rho/Rac/Cdc42 activator, please see Cat. #CN04.

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.
Rac and Cdc42 Activator (Cat. # 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. # BK005 and Figure 2) and Rac or Cdc42 activity assays by G-LISA (Cat. #BK125 and BK127 resp.) See Figure 1 for example of Cdc42 activation measured by the G-LISA assay. EGF is standardized in CN02 by measurement in units, thus 100 ng of EGF is 1 unit of CN02.

Rac/Cdc42 Activator II, epidermal growth factor (EGF) acts through a tyrosine kinase receptor (EGFR) to rapidly stimulate actin reorganization at the cell membrane resulting in membrane ruffling. This primary morphological response, which occurs within 1-10 minutes in Swiss 3T3 cells, has been shown to be mediated through activation of Rac (1). Biochemical assays that quantitate the amount of active (GTP-bound) small G-proteins have demonstrated that both Rac and Cdc42 are rapidly activated (within 30 seconds-2minutes) by EGF stimulation (2, 3). A later, secondary response to EGF treatment is the formation of actin stress fibers which is mediated through the activation of Rho (1). EGF is a useful tool in studying Rho signaling pathways, it should be noted, that EGF activates several other important signal transduction pathways including Ras/Raf/MAPK, JAK/STAT and PI3K/AKT and data should be interpreted accordingly.

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-LISA^TM Rac Activation Assay (Cat.BK125) and observed by ruffles and lamellipodia formation (see Figure 2B). 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-LISA^TM Cdc42 Activation Assay (Cat. BK127 see Figure 1).
Swiss 3T3 cells were serum starved (SS) for 16 h at 1% serum and 8 h with 0% serum and treated with CN01 (0.1, 0.5 and 1.0 units/ml for 1.5, 3.0, 6.0, 10 and 30 min). Cell lysates subjected to the Cdc42 G-LISA (BK127) 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 1.0 unit/ml the total activation was 2.05 fold or 105% over the controlled state at 1.5 min.
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). 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.