Category:Method/Sample/PL/Oxidized: Difference between revisions

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# Samples were applied to preconditioned (20mL of methanol and 20mL of water) C18 Sep-Pak cartridges (500mg, Waters, Millford, MA, USA).
# Samples were applied to preconditioned (20mL of methanol and 20mL of water) C18 Sep-Pak cartridges (500mg, Waters, Millford, MA, USA).
# Cartridge was washed with 20mL of water to exclude nonvolatile ions followed by 10mL of hexane to exclude cholesterols and neutral lipids.
# Cartridge was washed with 20mL of water to exclude nonvolatile ions followed by 10mL of hexane to exclude cholesterols and neutral lipids.
# Samples were eluted with 10mL of methanol to obtain oxidized phospholipids.
# Samples were eluted with 10mL of methyl formate to obtain oxidized fatty acids and 10mL of methanol to obtain oxidized phospholipids, subsequently.
# Lipid extracts were dried under a gentle stream of nitrogen, dissolved in 1mL of methanol, and stored at −80 ◦C until use.
# Lipid extracts were dried under a gentle stream of nitrogen, dissolved in 1mL of methanol, and stored at −80 ◦C until use.
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|
# メタノール 1mL で均質化し、氷上で抽出 1 h
# メタノール 1mL でホモジナイズ、氷上で1時間抽出
# 遠心分離 (7000 rpm, 4 ◦C, 5min).
# 遠心分離 (7000 rpm, 4 ◦C, 5min)  
# 上清を pH 3.0, 0.1N HCl　の水10倍量で希釈
# 上清を 0.1N HClでpH 3.0に調整した10倍量の水で希釈
# 条件設定済み (20mL of methanol and 20mL of water) のC18 Sep-Pak カートリッジ (500mg, Waters, Millford, MA, USA)に設定
# メタノール20mL 次いで水20mL で洗浄したC18 Sep-Pak カートリッジ (500mg, Waters, Millford, MA, USA)にサンプルを注入
# 20mL の水でカートリッジに残るイオンを洗浄、ヘキサン 10mL でコレステロールと中性脂質を除く
# 20mL の水でカートリッジに残るイオンを洗浄、ヘキサン 10mL でコレステロールと中性脂質を除く
# メタノール 10mL で酸化リン脂質を溶出
# ギ酸メチル　10mLで酸化脂肪酸を溶出、メタノール 10mL で酸化リン脂質を溶出
# 脂質をゆるやかな窒素ガスで乾燥、1mL メタノールに溶解、 −80 ◦C で保管
# 脂質をゆるやかな窒素ガスで乾燥、1mL メタノールに溶解、 −80 ◦C で保管
}}
}}

Revision as of 04:26, 20 October 2010

Oxidized phospholipids

  1. Samples were homogenized with 1mL methanol and extracted for 1 h at on ice.
  2. Homogenates were centrifuged (7000 rpm, 4 ◦C, 5min).
  3. Supernatant was diluted with 10 volumes of water adjusted to pH 3.0 with 0.1N HCl.
  4. Samples were applied to preconditioned (20mL of methanol and 20mL of water) C18 Sep-Pak cartridges (500mg, Waters, Millford, MA, USA).
  5. Cartridge was washed with 20mL of water to exclude nonvolatile ions followed by 10mL of hexane to exclude cholesterols and neutral lipids.
  6. Samples were eluted with 10mL of methyl formate to obtain oxidized fatty acids and 10mL of methanol to obtain oxidized phospholipids, subsequently.
  7. Lipid extracts were dried under a gentle stream of nitrogen, dissolved in 1mL of methanol, and stored at −80 ◦C until use.

  1. メタノール 1mL でホモジナイズ、氷上で1時間抽出
  2. 遠心分離 (7000 rpm, 4 ◦C, 5min)
  3. 上清を 0.1N HClでpH 3.0に調整した10倍量の水で希釈
  4. メタノール20mL 次いで水20mL で洗浄したC18 Sep-Pak カートリッジ (500mg, Waters, Millford, MA, USA)にサンプルを注入
  5. 20mL の水でカートリッジに残るイオンを洗浄、ヘキサン 10mL でコレステロールと中性脂質を除く
  6. ギ酸メチル　10mLで酸化脂肪酸を溶出、メタノール 10mL で酸化リン脂質を溶出
  7. 脂質をゆるやかな窒素ガスで乾燥、1mL メタノールに溶解、 −80 ◦C で保管

OxidizedMRMreverseLC.png 酸化脂質解析のための分子特異的測定方法
OxidizedReverseLC.png 逆相LCによるリン脂質・酸化リン脂質の分離
OxidizedMRMreverseLC.png MRMの特異性と逆相LCによる分離
OxidizedPC-MS2.png 酸化リン脂質のMS/MS解析と測定方法

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