MTORC1 Kinase Assay
From Bridges Lab Protocols
- 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
- 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