Category:Method/Sample/PL/Acidic PI - Revision history

Editor at 08:17, 5 November 2010

2010-11-05T08:17:10Z

← Older revision		Revision as of 17:17, 5 November 2010
Line 1:		Line 1:
	{{Taguchi/Header}}		{{Taguchi/Header}}
	=~~acidic~~ phospholipids (polyphosphoinositides)=		=Acidic phospholipids (polyphosphoinositides)=
	{{Twocolumn\|		{{Twocolumn\|
	# Cultured cells were scraped with 2 mL of ice-cold methanol, 100 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.		# Cultured cells were scraped with 2 mL of ice-cold methanol, 100 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.

Editor at 08:16, 5 November 2010

2010-11-05T08:16:54Z

← Older revision		Revision as of 17:16, 5 November 2010
Line 1:		Line 1:
			{{Taguchi/Header}}
	=acidic phospholipids (polyphosphoinositides)=		=acidic phospholipids (polyphosphoinositides)=
	{{Twocolumn\|		{{Twocolumn\|

Dbmanager at 05:51, 27 October 2010

2010-10-27T05:51:43Z

← Older revision		Revision as of 14:51, 27 October 2010
Line 4:		Line 4:
	# 2mL of 1 N HCl and 1mL of water were added to suspension. The solution was shaken well.		# 2mL of 1 N HCl and 1mL of water were added to suspension. The solution was shaken well.
	# 0.15 mL of 2 M NaCl and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.		# 0.15 mL of 2 M NaCl and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.
	# Samples were centrifuged at ~~1000g~~ for 5 min, and the lower layer was collected.		# Samples were centrifuged at 2000 rpm for 5 min, and the lower layer was collected.
	# After addition of 1.5 mL of methanol, each sample was applied to a DEAE-cellulose (Wako Pure Chemicals) column.		# After addition of 1.5 mL of methanol, each sample was applied to a DEAE-cellulose (Wako Pure Chemicals) column.
	# Column-bound lipids were washed with chloroform/methanol (1:1) (3 × 1 mL), and chloroform/methanol/28% aqueous ammonia/acetic acid (200:100:3:1) (3 × 1 mL) in series.		# Column-bound lipids were washed with chloroform/methanol (1:1) (3 × 1 mL), and chloroform/methanol/28% aqueous ammonia/acetic acid (200:100:3:1) (3 × 1 mL) in series.
Line 15:		Line 15:
	# 2N塩酸1mLと水1mLを添加し、よく撹拌する。		# 2N塩酸1mLと水1mLを添加し、よく撹拌する。
	# 2M 塩化ナトリウム水溶液、0.15mLとクロロホルム2mL を添加し、氷上で激しく撹拌する（３分）		# 2M 塩化ナトリウム水溶液、0.15mLとクロロホルム2mL を添加し、氷上で激しく撹拌する（３分）
	# ~~遠心（2000g~~ 5分）し、下層を分取する。		# 遠心（2000rpm 5分）し、下層を分取する。
	# 得られた抽出液にメタノール1.5mLを混和し、DEAEセルロースカラム(Wako Pure Chemicals)に注入する。		# 得られた抽出液にメタノール1.5mLを混和し、DEAEセルロースカラム(Wako Pure Chemicals)に注入する。
	# クロロホルム／メタノール(1:1) 3 x 1mL、次いでクロロホルム／メタノール／28%アンモニア水／氷酢酸 (200:100:3:1) 3 x 1mLでカラムを洗浄する。		# クロロホルム／メタノール(1:1) 3 x 1mL、次いでクロロホルム／メタノール／28%アンモニア水／氷酢酸 (200:100:3:1) 3 x 1mLでカラムを洗浄する。

Dbmanager at 05:37, 27 October 2010

2010-10-27T05:37:01Z

← Older revision		Revision as of 14:37, 27 October 2010
Line 13:		Line 13:
	\|		\|
	# 細胞を冷メタノール（2mL）に懸濁し、内標準　PIP1(16:0/16:0), PIP2(16:0/16:0), PIP3(16:0/16:0)　各2pmol/μLを50μL添加する。更に吸着を防御するためにPIP2(8:0/8:0) 1nmol/μLを2μL添加する。		# 細胞を冷メタノール（2mL）に懸濁し、内標準　PIP1(16:0/16:0), PIP2(16:0/16:0), PIP3(16:0/16:0)　各2pmol/μLを50μL添加する。更に吸着を防御するためにPIP2(8:0/8:0) 1nmol/μLを2μL添加する。
	# ~~2N塩酸1mLと水1mL　添加し、よく撹拌する。~~		# 2N塩酸1mLと水1mLを添加し、よく撹拌する。
	# 2M ~~塩化ナトリウム水溶液 0~~.15mLとクロロホルム2mL を添加し、氷上で激しく撹拌する（３分）		# 2M 塩化ナトリウム水溶液、0.15mLとクロロホルム2mL を添加し、氷上で激しく撹拌する（３分）
	# ~~遠心分離（2000g~~ 5分）し、下層を分取する。		# 遠心（2000g 5分）し、下層を分取する。
	# 得られた抽出液にメタノール1.5mLを混和し、DEAEセルロースカラム(Wako Pure Chemicals)に注入する。		# 得られた抽出液にメタノール1.5mLを混和し、DEAEセルロースカラム(Wako Pure Chemicals)に注入する。
	# クロロホルム／メタノール(1:1) 3 x 1mL、次いでクロロホルム／メタノール／28%アンモニア水／氷酢酸 (200:100:3:1) 3 x 1mLでカラムを洗浄する。		# クロロホルム／メタノール(1:1) 3 x 1mL、次いでクロロホルム／メタノール／28%アンモニア水／氷酢酸 (200:100:3:1) 3 x 1mLでカラムを洗浄する。

Dbmanager at 05:33, 27 October 2010

2010-10-27T05:33:56Z

← Older revision		Revision as of 14:33, 27 October 2010
Line 12:		Line 12:
	# The sample was redissolved in 0.05 mL of methanol/70% ethylamine (100:0.065), followed by addition of 0.03 mL of 1 M ammonium bicarbonate.		# The sample was redissolved in 0.05 mL of methanol/70% ethylamine (100:0.065), followed by addition of 0.03 mL of 1 M ammonium bicarbonate.
	\|		\|
	# 細胞を冷メタノール（2mL）に懸濁し、内標準　PIP1(16:0/16:0), PIP2(16:0/16:0), PIP3(16:0/16:0)　各2pmol/μLを50μL添加する。更に吸着を防御するためにPIP2(18:0/8:0) 1nmol/μLを2μL添加する。		# 細胞を冷メタノール（2mL）に懸濁し、内標準　PIP1(16:0/16:0), PIP2(16:0/16:0), PIP3(16:0/16:0)　各2pmol/μLを50μL添加する。更に吸着を防御するためにPIP2(8:0/8:0) 1nmol/μLを2μL添加する。
	# 2N塩酸1mLと水1mL　添加し、よく撹拌する。		# 2N塩酸1mLと水1mL　添加し、よく撹拌する。
	# 2M 塩化ナトリウム水溶液 0.15mLとクロロホルム2mL を添加し、氷上で激しく撹拌する（３分）		# 2M 塩化ナトリウム水溶液 0.15mLとクロロホルム2mL を添加し、氷上で激しく撹拌する（３分）

Dbmanager at 05:33, 27 October 2010

2010-10-27T05:33:02Z

← Older revision		Revision as of 14:33, 27 October 2010
Line 1:		Line 1:
	=acidic phospholipids (polyphosphoinositides)=		=acidic phospholipids (polyphosphoinositides)=
	{{Twocolumn\|		{{Twocolumn\|
	# Cultured cells were scraped with 2 mL of ice-cold methanol, 50 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.		# Cultured cells were scraped with 2 mL of ice-cold methanol, 100 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.
	# 2mL of 1 N HCl and 1mL of water were added to suspension. The solution was shaken well.		# 2mL of 1 N HCl and 1mL of water were added to suspension. The solution was shaken well.
	# 0.15 mL of 2 M NaCl and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.		# 0.15 mL of 2 M NaCl and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.
Line 12:		Line 12:
	# The sample was redissolved in 0.05 mL of methanol/70% ethylamine (100:0.065), followed by addition of 0.03 mL of 1 M ammonium bicarbonate.		# The sample was redissolved in 0.05 mL of methanol/70% ethylamine (100:0.065), followed by addition of 0.03 mL of 1 M ammonium bicarbonate.
	\|		\|
	# ~~細胞を冷メタノール懸濁液（2mL）に懸濁し、内標準　PIP1~~(16:0/16:0), PIP2(16:0/16:0), PIP3(16:0/16:0)　各2pmol/μLを50μL添加する。更に吸着を防御するためにPIP2(16:0) 1nmol/μLを2μL添加する。		# 細胞を冷メタノール（2mL）に懸濁し、内標準　PIP1(16:0/16:0), PIP2(16:0/16:0), PIP3(16:0/16:0)　各2pmol/μLを50μL添加する。更に吸着を防御するためにPIP2(18:0/8:0) 1nmol/μLを2μL添加する。
	# 2N塩酸1mLと水1mL　添加し、よく撹拌する。		# 2N塩酸1mLと水1mL　添加し、よく撹拌する。
	# 2M 塩化ナトリウム水溶液 0.15mLとクロロホルム2mL を添加し、氷上で激しく撹拌する（３分）		# 2M 塩化ナトリウム水溶液 0.15mLとクロロホルム2mL を添加し、氷上で激しく撹拌する（３分）

Dbmanager at 05:28, 27 October 2010

2010-10-27T05:28:26Z

← Older revision		Revision as of 14:28, 27 October 2010
Line 1:		Line 1:
	=acidic phospholipids (polyphosphoinositides)=		=acidic phospholipids (polyphosphoinositides)=
	{{Twocolumn\|		{{Twocolumn\|
	# Cultured cells ~~(3 to 6 × 106 per 100 mm plate)~~ were scraped with 2 mL of ice-cold methanol, 50 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.		# Cultured cells were scraped with 2 mL of ice-cold methanol, 50 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.
	# 2mL of 1 N HCl and 1mL of water were added to suspension. The solution was shaken well.		# 2mL of 1 N HCl and 1mL of water were added to suspension. The solution was shaken well.
	# 0.15 mL of 2 M NaCl and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.		# 0.15 mL of 2 M NaCl and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.
	# Samples were centrifuged at 1000g for 5 min, and the lower layer was collected.		# Samples were centrifuged at 1000g for 5 min, and the lower layer was collected.
	# After addition of 1.5 mL of methanol, each sample was applied to a DEAE-cellulose (Wako Pure Chemicals) column.		# After addition of 1.5 mL of methanol, each sample was applied to a DEAE-cellulose (Wako Pure Chemicals) column.
	# Column-bound lipids were washed with ~~chloroform (1 mL),~~ chloroform/methanol (1:1) (3 × 1 mL), and chloroform/methanol/28% aqueous ammonia/acetic acid (200:100:3:~~0.9~~) (3 × 1 mL) in series.		# Column-bound lipids were washed with chloroform/methanol (1:1) (3 × 1 mL), and chloroform/methanol/28% aqueous ammonia/acetic acid (200:100:3:1) (3 × 1 mL) in series.
	# Highly acidic lipids, including PIPs, LPA, and LPS were eluted with chloroform/methanol/HCl (6:6:1) (3 × 1 mL).		# Highly acidic lipids, including PIPs, LPA, and LPS were eluted with chloroform/methanol/HCl/water (12:12:1:1) (3 × 1 mL).
	# After addition of 1.5 mL of water and 0.113 mL of 2 M NaCl, the solution was vigorously shaken and centrifuged to collect the lower layer.		# After addition of 1.5 mL of water and 0.113 mL of 2 M NaCl, the solution was vigorously shaken and centrifuged to collect the lower layer.
	# After addition of 2 μL of 1 nmol/μL PIP2(8:0/8:0), each sample was dried under nitrogen gas.		# After addition of 2 μL of 1 nmol/μL PIP2(8:0/8:0), each sample was dried under nitrogen gas.
	# The sample was redissolved in 0.05 mL of methanol/70% ethylamine (100:0.065), followed by addition of 0.03 mL of 1 M ammonium bicarbonate ~~containing 0.2 mM phosphoric acid~~.		# The sample was redissolved in 0.05 mL of methanol/70% ethylamine (100:0.065), followed by addition of 0.03 mL of 1 M ammonium bicarbonate.
	\|		\|
	# ~~日本語~~		# 細胞を冷メタノール懸濁液（2mL）に懸濁し、内標準　PIP1(16:0/16:0), PIP2(16:0/16:0), PIP3(16:0/16:0)　各2pmol/μLを50μL添加する。更に吸着を防御するためにPIP2(16:0) 1nmol/μLを2μL添加する。
			# 2N塩酸1mLと水1mL　添加し、よく撹拌する。
			# 2M 塩化ナトリウム水溶液 0.15mLとクロロホルム2mL を添加し、氷上で激しく撹拌する（３分）
			# 遠心分離（2000g 5分）し、下層を分取する。
			# 得られた抽出液にメタノール1.5mLを混和し、DEAEセルロースカラム(Wako Pure Chemicals)に注入する。
			# クロロホルム／メタノール(1:1) 3 x 1mL、次いでクロロホルム／メタノール／28%アンモニア水／氷酢酸 (200:100:3:1) 3 x 1mLでカラムを洗浄する。
			# 酸性脂質をクロロホルム／メタノール／濃塩酸／水 (12:12:1:1) 3 x 1mLで溶出する。
			# 溶出液に水1.5mLと2M塩化ナトリウム水溶液 0.113mLを加え激しく撹拌し、遠心後下層を分取する。
			# 1nmol/μL PIP2(8:0/8:0) 2μLを加え、溶媒を窒素気流で留去する。
			# 得られた酸性脂質をメタノール/70%エチルアミン(100:0.065)0.05mLで再溶解後、1M炭酸水素アンモニウム水溶液0.03mLを混和し、試料とする。
	}}		}}

Dbmanager at 04:54, 27 October 2010

2010-10-27T04:54:45Z

← Older revision		Revision as of 13:54, 27 October 2010
Line 2:		Line 2:
	{{Twocolumn\|		{{Twocolumn\|
	# Cultured cells (3 to 6 × 106 per 100 mm plate) were scraped with 2 mL of ice-cold methanol, 50 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.		# Cultured cells (3 to 6 × 106 per 100 mm plate) were scraped with 2 mL of ice-cold methanol, 50 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.
	# 2mL of 1 N HCl and 1mL of water were added to suspension ~~and shaked~~ well.		# 2mL of 1 N HCl and 1mL of water were added to suspension. The solution was shaken well.
	# 0.15 mL of 2 M NaCl, and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.		# 0.15 mL of 2 M NaCl and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.
	# Samples were centrifuged at 1000g for 5 min, and the lower layer was collected.		# Samples were centrifuged at 1000g for 5 min, and the lower layer was collected.
	# After addition of 1.5 mL of methanol, each sample was applied to a DEAE-cellulose (Wako Pure Chemicals) column.		# After addition of 1.5 mL of methanol, each sample was applied to a DEAE-cellulose (Wako Pure Chemicals) column.

Dbmanager at 04:41, 27 October 2010

2010-10-27T04:41:07Z

← Older revision		Revision as of 13:41, 27 October 2010
Line 2:		Line 2:
	{{Twocolumn\|		{{Twocolumn\|
	# Cultured cells (3 to 6 × 106 per 100 mm plate) were scraped with 2 mL of ice-cold methanol, 50 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.		# Cultured cells (3 to 6 × 106 per 100 mm plate) were scraped with 2 mL of ice-cold methanol, 50 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.
	# 2mL of 1 N HCl, 0.15 mL of 2 M NaCl, and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.		# 2mL of 1 N HCl and 1mL of water were added to suspension and shaked well.
			# 0.15 mL of 2 M NaCl, and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.
	# Samples were centrifuged at 1000g for 5 min, and the lower layer was collected.		# Samples were centrifuged at 1000g for 5 min, and the lower layer was collected.
	# After addition of 1.5 mL of methanol, each sample was applied to a DEAE-cellulose (Wako Pure Chemicals) column.		# After addition of 1.5 mL of methanol, each sample was applied to a DEAE-cellulose (Wako Pure Chemicals) column.

Editor: New page: =acidic phospholipids (polyphosphoinositides)= {{Twocolumn| # Cultured cells (3 to 6 × 106 per 100 mm plate) were scraped with 2 mL of ice-cold methanol, 50 pmol of PIP1(16:0/16:0), PIP2(...

2010-10-10T01:28:05Z

New page: =acidic phospholipids (polyphosphoinositides)= {{Twocolumn| # Cultured cells (3 to 6 × 106 per 100 mm plate) were scraped with 2 mL of ice-cold methanol, 50 pmol of PIP1(16:0/16:0), PIP2(...

New page

=acidic phospholipids (polyphosphoinositides)=
{{Twocolumn|
# Cultured cells (3 to 6 × 106 per 100 mm plate) were scraped with 2 mL of ice-cold methanol, 50 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.
# 2mL of 1 N HCl, 0.15 mL of 2 M NaCl, and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.
# Samples were centrifuged at 1000g for 5 min, and the lower layer was collected.
# After addition of 1.5 mL of methanol, each sample was applied to a DEAE-cellulose (Wako Pure Chemicals) column.
# Column-bound lipids were washed with chloroform (1 mL), chloroform/methanol (1:1) (3 × 1 mL), and chloroform/methanol/28% aqueous ammonia/acetic acid (200:100:3:0.9) (3 × 1 mL) in series.
# Highly acidic lipids, including PIPs, LPA, and LPS were eluted with chloroform/methanol/HCl (6:6:1) (3 × 1 mL).
# After addition of 1.5 mL of water and 0.113 mL of 2 M NaCl, the solution was vigorously shaken and centrifuged to collect the lower layer.
# After addition of 2 μL of 1 nmol/μL PIP2(8:0/8:0), each sample was dried under nitrogen gas.
# The sample was redissolved in 0.05 mL of methanol/70% ethylamine (100:0.065), followed by addition of 0.03 mL of 1 M ammonium bicarbonate containing 0.2 mM phosphoric acid.
|
# 日本語

}}