MTORC1 Kinase Assay

Materials

• Cells treated as required.
• Purified GST-HA-S6K1 (purified from 293T cells)
• 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 (250 uM) and GST-S6K (200ng/rxn) to kinase buffer , 20 uL per reaction.
• Incubate at 30C for 30 min.
• Stop reaction with SDS Sample Buffer
• Detect phosphorylation by phospho-specific antibody western blotting.

see PMID 19200882