• Rapid, specific, irreversible activation of RhoA, -B, and -C proteins in live cells
• Direct activation of Rho
• Positive control for Rho activation in live cells
• Study the mechanisms underlying Rho-mediated events

CN03, part of the G-Switch™ line, is derived from the catalytic domain of bacterial CNF toxins and linked to a proprietary cell-penetrating moiety. It directly activates Rho GTPases by deamidating glutamine-63 in the Switch II region, converting it to glutamate and blocking GTPase activity, resulting in constitutive Rho activation. Produced in a bacterial expression system, CN03 is a 118 kDa His-tagged recombinant protein, supplied as 20 µg per vial.

Preparation & Use:

• Supplied as a lyophilized powder
• Reconstitute each tube in 200 µl sterile water to give 0.1 mg/ml of reagent
• Recommended working concentration: 1 µg/ml
• Each tube is sufficient for 20 ml of working-strength solution

Protein purity is assessed by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gel. Purity was determined to be >80%.

Biological activity of CN03 is demonstrated by its ability to robustly activate RhoA in live Swiss 3T3 cells. Treatment at 1 µg/ml produces optimal activation, approximately tenfold, within 1-2 hours as measured by a RhoA G-LISA assay BK124.

A protein containing a single point mutation that inactivates the catalytic domain of CN03 showed zero activity in a biological assay for Rho activation.

Responses to CN03 will vary depending on the cell line used.

• Activation timeline: typically 1-4 hours to activation peak
• Activation duration: irreversible activation, ≥ 4 hours
• Typical activation: 10-15 fold