• Detect activated (GTP-bound) Cdc42 protein in cell and tissue lysates
• Study Cdc42 signaling pathways

Kit contents (20-50 assays)

• P21 activated kinase1 protein (PAK)-PBD beads for affinity purification of active Cdc42 & Rac
• Cdc42-specific antibody to detect activated Cdc42 only
• His-Cdc42 control protein
• Cell lysis buffer-optimized for maintenance of GTP-bound Cdc42 & Rac
• Wash buffer-optimized for maintenance of GTP-bound Cdc42 & Rac
• Loading buffer: for control reactions
• STOP buffer: for control reactions
• GTPγS: for control reactions
• GDP: for control reactions
• Protease inhibitor cocktail
• DMSO: for protease resuspension

Equipment & materials required

• Cell lysates
• Cell scraper for harvesting cells
• Liquid nitrogen for snap freezing lysates
• Equipment for Western blot performance & analysis
• Microcentrifuge

The Cdc42 pull-down assay isolates active Cdc42/Rac (GTP-bound form) using the Cdc42/Rac-binding domain (PBD) of an effector protein immobilized on agarose beads. The bound Cdc42 is then detected and quantified by Western blotting using a Cdc42-specific antibody.

Key characteristics

• Semi-quantitative assay
• Detection sensitivity: 0.5 ng of activated Cdc42
• Detection specificity: Cdc42
• Input requirements: 200 µg to 1mg of cell or tissue lysate per assay
• Timeframe: Approximately 10 hours, typically performed over two days