MTORC1 Kinase Assay

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  • 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