• Detect activated (GTP-bound) RhoA protein in cell and tissue lysates
• Assay of choice when lysate quantity is limiting, e.g. primary cell cultures
• Study RhoA signaling pathways
• Screen for RhoA activation inhibitors

Kit contents (96 assays: strip-wells allow single or multiple assays per run)

• 96-well RhoA-GTP binding plate
• Anti-RhoA antibody
• Secondary detection HRP labeled antibody
• RhoA control protein (constitutively active RhoA)
• Cell lysis buffer- optimized for maintenance of GTP-bound GTPases
• Binding buffer
• Wash buffer
• Antigen-presenting buffer
• Antibody dilution buffer
• HRP chemiluminescent detection reagents A & B
• Precision Red advanced protein assay reagent
• Protease inhibitor cocktail
• Strip holder

Equipment & materials required

• Cell lysates
• Cell scraper for harvesting cells
• Concentrated sulfuric acid (1 ml)
• Liquid nitrogen for snap freezing lysates
• Orbital microplate shakers (min 200 rpm) one at 4°C and one at room temperature
• Multiwell pipette
• Microplate luminometer

Overview

The RhoA G-LISA® assay isolates active RhoA/B/C (GTP-bound form) using the Rho-binding domain (RBD) of an effector protein coupled to a 96-well strip plate. The bound RhoA is then detected and quantified using a luminescence ELISA-based method and a RhoA-specific antibody.

Key characteristics

• Quantitative assay
• Detection sensitivity: Linear between 10 pg – 1 ng of activated RhoA
• Detection specificity: RhoA
• Input requirements: 10 – 50 µg of cell or tissue lysate per assay
• Timeframe: Approximately 3 hours