Yeast Sch9 Phosphorylation Assay - Revision history

Davebrid at 13:16, 24 June 2010

2010-06-24T13:16:43Z

← Older revision		Revision as of 13:16, 24 June 2010
Line 20:		Line 20:
	# Incubate overnight at room temperature.		# Incubate overnight at room temperature.
	# Add lysis buffer and blot using HA antibodies.		# Add lysis buffer and blot using HA antibodies.


			==From Park et al==
			Log phase cells were resuspended in 1 ml ice cold 0.2 M NaOH containing 0.2 % β-
			mercaptoethanol (v/v) and incubated on ice for 10 min. Then 50 μl of trichloroacetic acid
			was added and samples were incubated on ice for 10 min. 1M Tris base was added, and
			samples were boiled in 2X SDS sample buffer. SDS-PAGE and transfer to nitrocellulose
			was performed using standard protocols. Proteins were blotted from a 6% gel and a 4-20% gel

Davebrid at 17:04, 2 February 2010

2010-02-02T17:04:18Z

← Older revision		Revision as of 17:04, 2 February 2010
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	==Materials==		==Materials==
	* NTCB dissolve to 7.5 mM in water (		* NTCB dissolve to 7.5 mM in water (1.68 mg/mL)
	* '''Urea Lysis Buffer''': 50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi		* '''Urea Lysis Buffer''': 50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi
	* '''PPi''': 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF.		* '''PPi''': 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF.

Davebrid: modified to a protocol

2010-02-02T16:53:17Z

modified to a protocol

← Older revision		Revision as of 16:53, 2 February 2010
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	~~from PMID 17560372~~		==Materials==
			* NTCB dissolve to 7.5 mM in water (
			* '''Urea Lysis Buffer''': 50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi
			* '''PPi''': 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF.

	~~Kinase Assay~~		==Protocol==
			# Grow cells to mid log phase
	~~PPi: 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease~~ inhibitor ~~cocktail and 1 mM PMSF~~.		# Dilute to OD = 0.2 in YPD with or without inhibitor treatments.
			# Incubate 1h at 24C in YPD
	~~Cultures were mixed with~~ TCA (final concentration 6%) ~~and put~~ on ice for at ~~least~~ 5 min ~~before cells were pelleted~~, ~~washed twice~~ with ~~cold~~ acetone, and ~~dried~~ in a speed-vac~~. Cell lysis was done in 100 μl of urea buffer (50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi) with glass beads in a bead beater with subsequent heating~~ for 10 min ~~to 65°C~~. ~~For NTCB cleavage 30 μl of 0.5 M CHES (pH 10.5) and 20 μl of NTCB (7.5 mM~~ in ~~H2O) were added and samples incubated over night at RT before 1 vol~~ of ~~2× sample buffer (+20 mM TCEP and 0.5× PPi) was added. Further analysis was done by SDS-PAGE and immunoblotting using anti-HA antibody 12CA5 or anti-T570-P antiserum~~.		# Add TCA to a final concentration of 6% (180 uL for 3 mL culture volume)
			# Place cells on ice for 5 min
	~~from PMID 19748353~~		# Spin cells at 5000 RPM for 5 min.
			# Wash with 1 mL Acetone, spin and wash with acetone again and aspirate acetone.
	~~Sch9 Phosphorylation Analyses~~		# Dry cells in a speed-vac for 30 min.
			# Resuspend in 150 uL of Urea Lysis Buffer.
	To analyze Sch9T570A-HA5 C-terminal phosphorylation, we used the chemical fragmentation analysis as described previously ([Urban et al., 2007] and [Wanke et al., 2008]). For quantifications of Sch9 phosphorylation, NTCB-cleaved extracts were separated by 7.5% SDS-PAGE followed by immunoblotting with ~~anti-HA antibody 12CA5. The anti-HA antibody was detected with far-red fluorescent Alexa Fluor 680 dye-labeled secondary anti-mouse antibody~~ (~~Invitrogen, A21057), and fluorescence intensity was measured using the Odyssey Infrared Imaging System (LI-COR~~).		# Lyse with a beadbeater (3x 30s).
			# Heat at 65C for 10 min.
	~~from PMID 18513215~~		# Spin 2 min at high in eppendorf centrifuge.
			# Remove 100 uL and add 30 uL of 0.5M CHES and 20 uL of NTCB. Save an untreated lysate sample as well.
	~~Sch9 and Ypk2 carboxy-terminal phosphorylation~~		# Incubate overnight at room temperature.
			# Add lysis buffer and blot using HA antibodies.
	~~To analyse Sch9-5HA C-terminal phosphorylation, TB50 cells containing plasmids pJU450 and pJU676 were grown in SC-Ura, -His, -Leu to mid-log phase, harvested and re-suspended in YPAD + 0.2% Gln~~ at ~~0.5 OD600. Cells were grown~~ for ~~60 min at 30°C prior to addition of medium containing rapamycin or caffeine and subsequent incubation for another 30~~ min. ~~Chemical fragmentation analysis was done as described (Urban et al., 2007). To analyse Ypk2 phosphorylation, MP8 cells were grown in YPD + 0.~~2~~% glutamine~~ at ~~30°C to an OD600 between 0~~.6 and 0.~~8, at which point rapamycin or caffeine was added to the indicated final concentration~~. ~~Cells were shaken for~~ an ~~additional 30 min and then harvested~~ as ~~described in Urban et al~~. ~~(2007), but without 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage~~. ~~Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membrane~~ and ~~immunoblotted with anti-~~HA ~~antibody or rabbit anti-phospho-T659 Ypk2 antiserum (this antiserum detects both Sch9 phosphorylated at T737 by TORC1 as well as Ypk2 phosphorylated at T659 by TORC2; R. Loewith, unpublished)~~.

Davebrid at 16:22, 15 January 2010

2010-01-15T16:22:42Z

← Older revision		Revision as of 16:22, 15 January 2010
Line 3:		Line 3:
	Kinase Assay		Kinase Assay

	~~TORC1 was purified from RL175~~-~~2d or RL176~~-~~1b cells treated~~ with ~~drug vehicle or 200 nM rapamycin for 30 min. Cells grown at 30°C in YPD~~ (~~250 ml per assay point~~) ~~to an OD600 of not, vert, similar1.0 were chilled~~ on ice~~, collected by centrifugation~~, washed with ~~H2O~~, ~~resuspended~~ in lysis buffer (~~1× PBS, 10%~~ [~~w/v~~] ~~glycerol~~, 0.5% ~~[v/v] Tween 20~~, PI, and PPi)~~, transferred to 2 ml screw-cap tubes half-filled~~ with glass beads (0.5 ~~mm), and disrupted in a Fast Prep machine at 4°C~~ (~~Bio101; 5× 30 s at max~~. ~~speed~~)~~. Crude lysates were cleared of debris with two 1000 × g spins~~ and ~~protein concentrations normalized as necessary. Extracts were precleared over CL-4B Sepharose before 7~~ μl of ~~IgG Sepharose~~ (~~GE Healthcare~~) ~~per assay point was~~ added and ~~the mix rotated for 90 min~~ at ~~4°C. Beads were collected in a column, washed with cold lysis buffer, and aliquotted to~~ 1~~.5 ml tubes. Kinase reactions were performed in kinase~~ buffer (~~1× PBS, 20% glycerol, 0.5% Tween~~ 20~~, 4~~ mM ~~MgCl2, 10 mM DTT, 2 μg/ml heparin,~~ and ~~PI [−EDTA]) in a final volume of 30 μl containing not, vert, similar350 ng of recombinant Sch9~~. ~~Assays were started with the addition of 100 μM ATP and 50 μCi [γ-32P]ATP, shaken for 20 min at 30°C, and terminated with the addition of 8 μl of~~ 5× ~~SDS-PAGE sample buffer~~. ~~Samples were heated to 95°C for 5 min before being separated~~ by SDS-PAGE~~, stained with Coomassie,~~ and ~~analyzed~~ using ~~a BioRad Molecular Imager~~.		PPi: 10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenylphosphate, 10 mM Na2P2O4, and 10 mM β-glycerophosphate; PI: 1× Roche protease inhibitor cocktail and 1 mM PMSF.

			Cultures were mixed with TCA (final concentration 6%) and put on ice for at least 5 min before cells were pelleted, washed twice with cold acetone, and dried in a speed-vac. Cell lysis was done in 100 μl of urea buffer (50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS, 1 mM PMSF, and 0.5× PPi) with glass beads in a bead beater with subsequent heating for 10 min to 65°C. For NTCB cleavage 30 μl of 0.5 M CHES (pH 10.5) and 20 μl of NTCB (7.5 mM in H2O) were added and samples incubated over night at RT before 1 vol of 2× sample buffer (+20 mM TCEP and 0.5× PPi) was added. Further analysis was done by SDS-PAGE and immunoblotting using anti-HA antibody 12CA5 or anti-T570-P antiserum.

	from PMID 19748353		from PMID 19748353

Davebrid at 16:17, 15 January 2010

2010-01-15T16:17:03Z

← Older revision		Revision as of 16:17, 15 January 2010
Line 1:		Line 1:
	from PMID 17560372		from PMID 17560372

	Kinase Assay		Kinase Assay

	TORC1 was purified from RL175-2d or RL176-1b cells treated with drug vehicle or 200 nM rapamycin for 30 min. Cells grown at 30°C in YPD (250 ml per assay point) to an OD600 of not, vert, similar1.0 were chilled on ice, collected by centrifugation, washed with H2O, resuspended in lysis buffer (1× PBS, 10% [w/v] glycerol, 0.5% [v/v] Tween 20, PI, and PPi), transferred to 2 ml screw-cap tubes half-filled with glass beads (0.5 mm), and disrupted in a Fast Prep machine at 4°C (Bio101; 5× 30 s at max. speed). Crude lysates were cleared of debris with two 1000 × g spins and protein concentrations normalized as necessary. Extracts were precleared over CL-4B Sepharose before 7 μl of IgG Sepharose (GE Healthcare) per assay point was added and the mix rotated for 90 min at 4°C. Beads were collected in a column, washed with cold lysis buffer, and aliquotted to 1.5 ml tubes. Kinase reactions were performed in kinase buffer (1× PBS, 20% glycerol, 0.5% Tween 20, 4 mM MgCl2, 10 mM DTT, 2 μg/ml heparin, and PI [−EDTA]) in a final volume of 30 μl containing not, vert, similar350 ng of recombinant Sch9. Assays were started with the addition of 100 μM ATP and 50 μCi [γ-32P]ATP, shaken for 20 min at 30°C, and terminated with the addition of 8 μl of 5× SDS-PAGE sample buffer. Samples were heated to 95°C for 5 min before being separated by SDS-PAGE, stained with Coomassie, and analyzed using a BioRad Molecular Imager.		TORC1 was purified from RL175-2d or RL176-1b cells treated with drug vehicle or 200 nM rapamycin for 30 min. Cells grown at 30°C in YPD (250 ml per assay point) to an OD600 of not, vert, similar1.0 were chilled on ice, collected by centrifugation, washed with H2O, resuspended in lysis buffer (1× PBS, 10% [w/v] glycerol, 0.5% [v/v] Tween 20, PI, and PPi), transferred to 2 ml screw-cap tubes half-filled with glass beads (0.5 mm), and disrupted in a Fast Prep machine at 4°C (Bio101; 5× 30 s at max. speed). Crude lysates were cleared of debris with two 1000 × g spins and protein concentrations normalized as necessary. Extracts were precleared over CL-4B Sepharose before 7 μl of IgG Sepharose (GE Healthcare) per assay point was added and the mix rotated for 90 min at 4°C. Beads were collected in a column, washed with cold lysis buffer, and aliquotted to 1.5 ml tubes. Kinase reactions were performed in kinase buffer (1× PBS, 20% glycerol, 0.5% Tween 20, 4 mM MgCl2, 10 mM DTT, 2 μg/ml heparin, and PI [−EDTA]) in a final volume of 30 μl containing not, vert, similar350 ng of recombinant Sch9. Assays were started with the addition of 100 μM ATP and 50 μCi [γ-32P]ATP, shaken for 20 min at 30°C, and terminated with the addition of 8 μl of 5× SDS-PAGE sample buffer. Samples were heated to 95°C for 5 min before being separated by SDS-PAGE, stained with Coomassie, and analyzed using a BioRad Molecular Imager.
Line 7:		Line 7:
	from PMID 19748353		from PMID 19748353

	Sch9 Phosphorylation Analyses		Sch9 Phosphorylation Analyses

	To analyze Sch9T570A-HA5 C-terminal phosphorylation, we used the chemical fragmentation analysis as described previously ([Urban et al., 2007] and [Wanke et al., 2008]). For quantifications of Sch9 phosphorylation, NTCB-cleaved extracts were separated by 7.5% SDS-PAGE followed by immunoblotting with anti-HA antibody 12CA5. The anti-HA antibody was detected with far-red fluorescent Alexa Fluor 680 dye-labeled secondary anti-mouse antibody (Invitrogen, A21057), and fluorescence intensity was measured using the Odyssey Infrared Imaging System (LI-COR).		To analyze Sch9T570A-HA5 C-terminal phosphorylation, we used the chemical fragmentation analysis as described previously ([Urban et al., 2007] and [Wanke et al., 2008]). For quantifications of Sch9 phosphorylation, NTCB-cleaved extracts were separated by 7.5% SDS-PAGE followed by immunoblotting with anti-HA antibody 12CA5. The anti-HA antibody was detected with far-red fluorescent Alexa Fluor 680 dye-labeled secondary anti-mouse antibody (Invitrogen, A21057), and fluorescence intensity was measured using the Odyssey Infrared Imaging System (LI-COR).

Davebrid: Created page with 'from PMID 17560372 Kinase Assay TORC1 was purified from RL175-2d or RL176-1b cells treated with drug vehicle or 200 nM rapamycin for 30 min. Cells grown at 30°C in YPD (250 ...'

2010-01-15T16:16:45Z

Created page with 'from PMID 17560372 Kinase Assay TORC1 was purified from RL175-2d or RL176-1b cells treated with drug vehicle or 200 nM rapamycin for 30 min. Cells grown at 30°C in YPD (250 ...'

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from PMID 17560372

Kinase Assay

TORC1 was purified from RL175-2d or RL176-1b cells treated with drug vehicle or 200 nM rapamycin for 30 min. Cells grown at 30°C in YPD (250 ml per assay point) to an OD600 of not, vert, similar1.0 were chilled on ice, collected by centrifugation, washed with H2O, resuspended in lysis buffer (1× PBS, 10% [w/v] glycerol, 0.5% [v/v] Tween 20, PI, and PPi), transferred to 2 ml screw-cap tubes half-filled with glass beads (0.5 mm), and disrupted in a Fast Prep machine at 4°C (Bio101; 5× 30 s at max. speed). Crude lysates were cleared of debris with two 1000 × g spins and protein concentrations normalized as necessary. Extracts were precleared over CL-4B Sepharose before 7 μl of IgG Sepharose (GE Healthcare) per assay point was added and the mix rotated for 90 min at 4°C. Beads were collected in a column, washed with cold lysis buffer, and aliquotted to 1.5 ml tubes. Kinase reactions were performed in kinase buffer (1× PBS, 20% glycerol, 0.5% Tween 20, 4 mM MgCl2, 10 mM DTT, 2 μg/ml heparin, and PI [−EDTA]) in a final volume of 30 μl containing not, vert, similar350 ng of recombinant Sch9. Assays were started with the addition of 100 μM ATP and 50 μCi [γ-32P]ATP, shaken for 20 min at 30°C, and terminated with the addition of 8 μl of 5× SDS-PAGE sample buffer. Samples were heated to 95°C for 5 min before being separated by SDS-PAGE, stained with Coomassie, and analyzed using a BioRad Molecular Imager.

from PMID 19748353

Sch9 Phosphorylation Analyses

To analyze Sch9T570A-HA5 C-terminal phosphorylation, we used the chemical fragmentation analysis as described previously ([Urban et al., 2007] and [Wanke et al., 2008]). For quantifications of Sch9 phosphorylation, NTCB-cleaved extracts were separated by 7.5% SDS-PAGE followed by immunoblotting with anti-HA antibody 12CA5. The anti-HA antibody was detected with far-red fluorescent Alexa Fluor 680 dye-labeled secondary anti-mouse antibody (Invitrogen, A21057), and fluorescence intensity was measured using the Odyssey Infrared Imaging System (LI-COR).

from PMID 18513215

Sch9 and Ypk2 carboxy-terminal phosphorylation

To analyse Sch9-5HA C-terminal phosphorylation, TB50 cells containing plasmids pJU450 and pJU676 were grown in SC-Ura, -His, -Leu to mid-log phase, harvested and re-suspended in YPAD + 0.2% Gln at 0.5 OD600. Cells were grown for 60 min at 30°C prior to addition of medium containing rapamycin or caffeine and subsequent incubation for another 30 min. Chemical fragmentation analysis was done as described (Urban et al., 2007). To analyse Ypk2 phosphorylation, MP8 cells were grown in YPD + 0.2% glutamine at 30°C to an OD600 between 0.6 and 0.8, at which point rapamycin or caffeine was added to the indicated final concentration. Cells were shaken for an additional 30 min and then harvested as described in Urban et al. (2007), but without 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted with anti-HA antibody or rabbit anti-phospho-T659 Ypk2 antiserum (this antiserum detects both Sch9 phosphorylated at T737 by TORC1 as well as Ypk2 phosphorylated at T659 by TORC2; R. Loewith, unpublished).