MTORC1 Kinase Assay
Revision as of 17:33, 26 August 2009 by Davebridges (Talk | contribs) (Created page with '==Materials== *Cells treated as required. *Purified GST-HA-S6K1 (purified from 293T cells) *mTOR antibody (Santa Cruz) *Protein A/G Beads *CHAPS Lysis Buffer *[[mTORC1 Kinase...')
Revision as of 17:33, 26 August 2009 by Davebridges (Talk | contribs) (Created page with '==Materials== *Cells treated as required. *Purified GST-HA-S6K1 (purified from 293T cells) *mTOR antibody (Santa Cruz) *Protein A/G Beads *CHAPS Lysis Buffer *[[mTORC1 Kinase...')
Materials
- Cells treated as required.
- Purified GST-HA-S6K1 (purified from 293T cells)
- mTOR antibody (Santa Cruz)
- Protein A/G Beads
- CHAPS Lysis Buffer
- mTORC1 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 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.