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MTORC1 Kinase Assay

131 bytes added, 18:15, 26 August 2009
added substrate and ATP separately
==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
*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 (250 uM) and to [[TORC1 Kinase Buffer]] at 500uM*Add 10 uL GST-S6K (200ng/rxn) S6K1 to kinase buffer , 20 beads*Add 10 uL per reaction.[[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.