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...')

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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...')

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Materials

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.