• Detect activated (GTP-bound) Arf1 protein in cell and tissue lysates
• Study Arf1 signaling pathways

Kit contents (20 assays)

• GGA3-PBD beads for affinity purification of active Arf1 & Arf6
• Arf1-specific antibody to detect activated Arf1 only
• His-Arf1 control protein
• Cell lysis buffer-optimized for maintenance of GTP-bound GTPases
• Wash buffer-optimized for maintenance of GTP-bound GTPases
• 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 Arf1 pull-down assay isolates active Arf1 (GTP-bound form) using the Arf1/6-binding domain (PBD) of an effector protein immobilized on agarose beads. The bound Arf1 is then detected and quantified by Western blotting using an Arf1-specific antibody.

Key characteristics

• Semi-quantitative assay
• Detection sensitivity: 1 ng of activated Arf1
• Detection specificity: Arf1
• Input requirements: 200 µg to 1mg of cell or tissue lysate per assay
• Timeframe: Approximately 10 hours, typically performed over two days