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Separation of
phospholipid subclasses |
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Dry sample. Dissolve in 2 ml anhydrous
diethyl ether. |
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Add 0.5 ml of 0.05 m K2HPO4
pH 7.0 plus 10 IU phospholipase C (3 ml). |
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Vortex. Leave 30 min at room temperature. |
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Evaporate the ether
in a N2 stream. |
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Add 1 ml chloroform/methanol
(2:1). |
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Vortex. Extract
thrice with 0.5 ml chloroform. |
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Dry the chloroform
phase. Dissolve the residue in 0.3 ml benzoic acid. |
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Add 2 mg benzoic anhydride and 0.8
mg 4-dimethyl aminopyridine. |
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Vortex. Leave 60
min at room temperature. |
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Add 1 ml concentrated
ammonia (on ice!!!). |
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Vortex. Extract
four times with 0.5 ml n-hexane. |
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Dry. Separate by
t.l.c. using the system benzene/n-hexane/diethy ether (50:45:1). |
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Approximate Rf values:
acyl species, ~0.21-0.29; alkyl species, ~0.35-0.39; alkenyl
species, ~0.40-0.45). |
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Back
to Protocols |
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