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

Kit contents (20-80 assays)

• Rhotekin-PBD beads for affinity purification of active RhoA, B & C
• RhoA-specific antibody to detect activated RhoA only
• His-RhoA control protein
• Cell lysis buffer-optimized for maintenance of GTP-bound Ras
• Wash buffer-optimized for maintenance of GTP-bound Ras
• 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 RhoA pull-down assay isolates active RhoA (GTP-bound form) using the RhoA/B/C-binding domain (RBD) of an effector protein immobilized on agarose beads. The bound RhoA is then detected and quantified by Western blotting using a RhoA-specific antibody.

Key characteristics

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