• Identify Guanine nucleotide exchange (GEF) proteins for Cdc42, Rac1 or RhoA proteins: in vitro assay using purified proteins, not suitable for use with cell lysates
• Biochemical characterization of GEF activity
• Identify GEF inhibitors in a high-throughput screen format

Kit contents (20 - 130 assays per GTPase)

• Exchange buffer
• Cdc42-His protein
• RhoA-His protein
• Rac1-His protein
• hDbs-His protein
• 384-well plate
• 96-well plate

Equipment & materials required

• Temperature regulated fluorimeter (ex: 485 nm ±20 nm; em 535 nm ±20 nm)

This fluorescence-based assay leverages the spectral difference between free and bound fluorescent guanine nucleotide analogs to monitor small GTPase nucleotide exchange. Upon binding to GTPases, fluorescence intensity increases sharply, enabling detection of GEF activity for known or novel proteins.

Key characteristics

• Detection method: Fluorescence-based assay: (ex: 485 nm em: 535 nm)
• Timeframe: Approximately 2 hours