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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...'
==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.
*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.
