Revision as of 08:29, 26 October 2010

Acidic phospholipids （PA, LPA, PS, LPS, PI）

Cultured cells were scraped with 2 mL of ice-cold butanol, and 50 pmol of LPA(17:0) and PA(14:0/14:0) were added as an internal standard.
2 mL of PBS was added to suspension, followed by vigorous shaking for 3 min while cooling with ice.
Samples were centrifuged at 2000g for 5 min, and the upper layer was collected.
After the addition of 1 mL of methanol and 1 mL of chloroform, each sample was applied to a DEAE-cellulose column.
Column-bound lipids were washed with chloroform/methanol (1:1) (3 × 1 mL).
Acidic lipids, containing PA, PS, and PI, and their lyso forms, were eluted with chloroform/methanol/28% aqueous ammonia/acetic acid (200:100:10:6.7) (3× 1 mL).
After removing organic solvent from each eluate under nitrogen gas, 0.05 mL of water and 0.1 mL of 2-propanol were added to about 0.05 mL of residue.

細胞の1-ブタノール懸濁液　2mLを調製し、内標準　LPA(17:0), PA(14:0/14:0)など各50 pmolを添加する
PBS　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、上層を分取する
ブタノール抽出液にメタノール1mLとクロロホルム1mLを混合したものを、DEAEセルロースにかける
カラムに結合した中性脂質をクロロホルム／メタノール(1:1) 3 x 1mLで洗浄する
クロロホルム／メタノール／28%アンモニア水／氷酢酸 (200:100:10:6.7) 3 x 1mLで酸性脂質(PA, PS, PIとそれぞれのリゾ体)を溶出する
溶出液を窒素気流で留去後、残存した水溶液約0.05mLに水0.05mLと2-プロパノール0.1mLを混和し試料とする

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@@ Line 15: / Line 15: @@
 # カラムに結合した中性脂質をクロロホルム／メタノール(1:1) 3 x 1mLで洗浄する
 # クロロホルム／メタノール／28%アンモニア水／氷酢酸 (200:100:10:6.7) 3 x 1mLで酸性脂質(PA, PS, PIとそれぞれのリゾ体)を溶出する
-# 溶出液の溶媒を窒素気流で留去後、残存した水溶液約0.05mLに水0.05mLと2-プロパノール0.1mLを混和し試料とする
+# 溶出液を窒素気流で留去後、残存した水溶液約0.05mLに水0.05mLと2-プロパノール0.1mLを混和し試料とする
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