Category:Method/Sample/PL/Acidic: Difference between revisions
(New page: =Acidic phospholipids （PA, LPA, PS, LPS, PI）= {{Twocolumn| # Cultured cells (3 to 6 × 106 per 100 mm plate) were scraped with 2 mL of ice-cold butanol, and 50 pmol of LPA(17:0) and PA...) |
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=Acidic phospholipids （PA, LPA, PS, LPS, PI）= | =Acidic phospholipids （PA, LPA, PS, LPS, PI）= | ||
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# Cultured cells | # 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 | # 2 mL of PBS was added to suspension, followed by vigorous shaking for 3 min while cooling with ice. | ||
# Samples were centrifuged at | # 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. | # 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 | # 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). | # 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. | # 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. | ||
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# | # 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セルロースカラムにかける | |||
# 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 | |||
}} | }} | ||
Latest revision as of 08:17, 5 November 2010
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Acidic phospholipids （PA, LPA, PS, LPS, PI）
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Pages in category "Method/Sample/PL/Acidic"
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