MTORC1 Kinase Assay

Materials

• Cells treated as required.
• Purified GST-HA-S6K1 (purified from 293T cells). Diluted in TORC1 Kinase Buffer to 200ng/10uL.
• mTOR antibody (Santa Cruz)
• Protein A/G Beads
• CHAPS Lysis Buffer
• TORC1 Kinase Buffer

Protocol

• Treat cells are required.
• Lyse cells for 20 min (for 293T cells) with CHAPS Lysis Buffer
• Centrifuge Lysates for 10 min at 14000 RPM in eppendorf centrifuge.
• Add lysate + 10 uL anti-mTOR antibody to a fresh tube and incubate end over end for 1h at 4C
• Add 15 uL Protein A/G Beads to tubes and incubate end over end for 1h at 4C
• Spin down beads for 15s at 14 000 RPM and aspirate with a fine-tipped (or crushed tip) pipet tip
• Wash beads 2x with Lysis Buffer and 1x with TORC1 Kinase Buffer (-ATP)
• Add ATP to TORC1 Kinase Buffer at 500uM
• Add 10 uL GST-S6K1 to beads
• Add 10 uL TORC1 Kinase Buffer with ATP and mix
• Incubate at 30C for 30 min, with occasional mixing.
• Stop reaction with 20 uL 2X SDS Sample Buffer.
• Detect phosphorylation by phospho-specific antibody western blotting.

see PMID 19200882