Difference between revisions of "MTORC1 Kinase Assay"

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(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...')
 
(added substrate and ATP separately)
 
(2 intermediate revisions by the same user not shown)
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==Materials==
 
==Materials==
 
*Cells treated as required.
 
*Cells treated as required.
*Purified GST-HA-S6K1 (purified from 293T cells)
+
*Purified GST-HA-S6K1 (purified from 293T cells).  Diluted in [[TORC1 Kinase Buffer]] to 200ng/10uL.
 
*mTOR antibody (Santa Cruz)
 
*mTOR antibody (Santa Cruz)
 
*Protein A/G Beads
 
*Protein A/G Beads
 
*[[CHAPS Lysis Buffer]]
 
*[[CHAPS Lysis Buffer]]
*[[mTORC1 Kinase Buffer]]
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*[[TORC1 Kinase Buffer]]
  
 
==Protocol==
 
==Protocol==
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*Add 15 uL Protein A/G Beads to tubes 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
 
*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)
+
*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.
+
*Add ATP to [[TORC1 Kinase Buffer]] at 500uM
*Incubate at 30C for 30 min.
+
*Add 10 uL GST-S6K1 to beads
*Stop reaction with SDS Sample Buffer
+
*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.
 
*Detect phosphorylation by phospho-specific antibody western blotting.
 +
 +
see PMID 19200882
 +
[[ Category:Buffer ]]
 +
[[ Category:Kinase Assay]]
 +
[[ Category:mTOR ]]

Latest revision as of 18:15, 26 August 2009

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 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