Category:Method/Sample/PL/Acidic

Top Lipid Class Method Symbols

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

  1. 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. 2 mL of PBS was added to suspension, followed by vigorous shaking for 3 min while cooling with ice.
  3. Samples were centrifuged at 2000g for 5 min, and the upper layer was collected.
  4. After the addition of 1 mL of methanol and 1 mL of chloroform, each sample was applied to a DEAE-cellulose column.
  5. Column-bound lipids were washed with chloroform/methanol (1:1) (3 × 1 mL).
  6. 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).
  7. 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.

  1. 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
  2. PBS 2mL を添加し、氷上で激しく撹拌する(3分)
  3. 遠心分離(2000g 5分)し、上層を分取する
  4. 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セルロースカラムにかける
  5. 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
  6. 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
  7. 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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