MTORC1 Kinase Assay - Revision history

Davebrid: added substrate and ATP separately

2009-08-26T18:15:27Z

added substrate and ATP separately

← Older revision		Revision as of 18:15, 26 August 2009
Line 1:		Line 1:
	==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
Line 15:		Line 15:
	*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 [[TORC1 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.

Davebrid at 17:34, 26 August 2009

2009-08-26T17:34:24Z

← Older revision		Revision as of 17:34, 26 August 2009
Line 5:		Line 5:
	*Protein A/G Beads		*Protein A/G Beads
	*[[CHAPS Lysis Buffer]]		*[[CHAPS Lysis Buffer]]
	*[[~~mTORC1~~ Kinase 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 (250 uM) and GST-S6K (200ng/rxn) to kinase buffer , 20 uL per reaction.		*Add ATP (250 uM) and GST-S6K (200ng/rxn) to kinase buffer , 20 uL per reaction.
	*Incubate at 30C for 30 min.		*Incubate at 30C for 30 min.

Davebrid at 17:33, 26 August 2009

2009-08-26T17:33:47Z

← Older revision		Revision as of 17:33, 26 August 2009
Line 19:		Line 19:
	*Stop reaction with SDS Sample Buffer		*Stop reaction with 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 ]]

Davebrid: 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...'

2009-08-26T17:33:16Z

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...'

New page

==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.

MTORC1 Kinase Assay - Revision history

Davebrid: added substrate and ATP separately

Davebrid at 17:34, 26 August 2009

Davebrid at 17:33, 26 August 2009

Davebrid: 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...'

Davebrid: 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...'