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

Kit contents (20-50 assays)

• Raf-RBD beads for affinity purification of active Ras
• Anti pan Ras antibody to detect activated Ras isotypes (H-, K-, and N). End user isotype specific antibody can be used for Ras specific isotype detection
• His-H-Ras 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 Ras pull-down assay isolates active Ras (GTP-bound form) using the Ras-binding domain (RBD) of an effector protein immobilized on agarose beads. The bound Ras is then detected and quantified by Western blotting.

Key characteristics

• Semi-quantitative assay
• Detection sensitivity: 1 ng of activated Ras
• Detection specificity: N-Ras, H-Ras, K-Ras (can be adapted to specific Ras isotypes)
• Input requirements: 200 µg to 1mg of cell or tissue lysate per assay
• Timeframe: Approximately 10 hours, typically performed over two days