1)
extract lipids by the Bligh and Dyer method as
follows:
a)
suspend cell pellet in 0.4 ml of buffer and add to a
13 x 100 mm glass tube containing 1.5 ml of
CHCl3/MeOH (1:2) and vortex
b) add 0.5 ml
of CHCl3 and vortex
c) add 0.5 ml
of H2O and vortex
d) centrifuge
to separate phases and retain the lower phase (either
discard the upper phase by aspiration or transfer the
lower phase to a new 13 x 100 mm tube)
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2)
concentrate the lower phase by evaporating
CHCl3 with N2 gas or by using speed
vac. concentrator
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3)
construct a standard curve using 0-300 pmoles of natural
ceramide from Avanti Polar Lipids and evaporate
CHCl3
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4) add 20
ml
of b-OG/DOPG
(7.5%:25mM) mixed micelles to the lipids in the
experimental samples and to the ceramide standards and
vortex vigorously
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5)
incubate for 30 min at 37° C and repeat
vortexing
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6) add 70
ml
of reaction mixture containing the following: 50
ml
of 2x buffer, 19.4 ml
of dilution buffer, 0.2 ml
of 1M DTT and 3-5 mg
of a DG kinase membrane preparation (the non-lyophilized
enzyme from Calbiochem gives best results)
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7) add 10
ml
of 10 mM [g-32P]-ATP
(2 mCi/rxn)
to each tube and allow rxn to proceed for 30 min at room
temp.
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8) stop
rxn by the addition of 1.5 ml of CHCl3/MeOH
(1:2)
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9) add
0.3 ml of H2O and vortex
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10) add
0.5 ml of CHCl3 and vortex
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11) add
0.5 ml of 1% HClO4 and vortex
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12)
centrifuge to separate phases and retain lower
phase
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13) dry
lower phase as previously described and suspend lipids in
30-40 ml
of CHCl3/MeOH (4:1)
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14)
chromatograph 1/2 of total lipid on a silica gel 60 thin
layer chromatography plate using a solvent system of
CHCl3/acetone/MeOH/HOAc/H2O
(10:4:3:2:1)
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15) the
[32P]-ceramide (ceramide 1-phosphate)
is detected by using either a phosphorimager or by X-ray
film
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