MTORC1 Kinase Assay: Difference between revisions
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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]] | ||
*[[ | *[[TORC1 Kinase Buffer]] | ||
==Protocol== | ==Protocol== | ||
| Line 14: | Line 14: | ||
*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 | *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. | ||
Latest revision as of 18:15, 26 August 2009
Materials
- 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
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