MTORC1 Kinase Assay: Difference between revisions
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 |
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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== | ||
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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 | *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 ]] | |||