Lipid extraction from Arabidopsis rosette leaves
Sampling Table
Sample name (date):
Sample No.

ExampleFresh weight (g)1.0

H2O from material(ml) ––– (W)（生重量 x0.9）
2-Propanol(ml)Homogenate (ml)Methanol
Chloroform (1)H2O

Pellet washing (ml)Methanol
Chloroform (2)H20CHCl3

layer = (1) + (2)H20

Empty vessel (g)Total lipid extracts(mg)

No. 2

No. 3

555555555555２２２２

１１１

１０．８

０．８

０．８

０．８

６６６６６６６６0.9

3.1=4.0-(W)

CombinedNo. 1

Day 1
Place weighed rosette leaves (1 g) in a 50 ml screw-capped glass tube.
Flash 5 ml hot 2-propanol (preheated in 80˚C water bath) over the rosetteleaves using a 10-ml silicon-rubber-capped glass pipette so that all leaves are
immediately immersed in 2-propanol. The resultant samples (in a 50-ml tube)
should be immediately put into the 80˚C water bath.
↓ 5 min, 80˚C
Chilled on ice.
Add methanol, chloroform, and water as calculated from the Sampling Table.
↓Homogenize samples on ice to homogeneity, using a Polytron (max. speed),↓3000 rpm x 15 min (r.t.)
Recover the supernatant (A) into a fresh 50-ml screw-capped glass tube bydecantation (if the pellet is soft, remove by pipetting).
↓Resuspend the pellet in methanol, chloroform and water, as listed in theSampling Table, and mix well using a vortex mixer.
↓ 3000 rpm x 15 min (r.t.)
Recover the supernatant (B) and combine with the supernatant A.
↓Add 6 ml chloroform and 6 ml H2O to the combined supernatant and mix
vigorously using a vortex mixer.
↓3000 rpm x 15 min (r.t.)
Recover the lower (chloroform) layer into a fresh 50-ml screw-capped glass
tube with an equal volume of 0.9% KCl.
↓3000 rpm x 15 min (r.t)
Recover the lower (chloroform) layer*1 into a 20 ml weighed pear-shaped flask,and evaporate the solvent on a rotary evaporator*2.
*1Special attention should be paid not to recover any material floating in theintermediate phase.
2* If you see water droplets on the bottom of flask, add a few ml of ethanol
and evaporate again.
↓Dry up the residual solvent in a vacuum desiccator and determine the lipid dry

weight.
↓Add chloroform to make a 10 mg/ml lipid solution while placing the flask on ice
to prevent solvent evaporation.
↓Transfer the lipid solution into a 1-ml screw-capped sample tube*3.
*3Because the cap of the tube is so small, special attention should be paid
to ensure that the cap is screwed tightly on the tube. Each cap should
then be sealed with parafilm and stored at -30 ˚C. If you store the sample
for more than one week, the antioxidant butylhydroxyltoluene (BHT)
should be added at a final concentration of 10 µg/ml.

Day 2
Separation of lipids
chromatography

bytwo-dimensional

silicagelthin-layer

Using a 100-µl glass micro syringe, streak 100 µl of the lipid extract (1 mg lipidequivalent) on a 1-cm line marked in the 2-cm-from-right and 2-cm-from-bottom
position of a TLC plate (20 x 20 cm, Silica gel 60, Merck 1.05721.) as shown
below.
A view before the second
When streaking lipids

TLC development

1st

2nd

2nd

1st

Estimated weight of lipid classes in 1 mg total lipid extracts:
Pigments
0.59 mg
Glycolipids
0.2 mgNeutral phospholipids0.12 mg
Acidic lipids0.06 mg
1st dimension：
Acetone/Benzene/Methanol/water = 8:3:2:1（by volume)↓Air-dry the plate (you may use warm air from a hair dryer).
↓
2nd dimension：
Chloroform/Methanol/Acetic acid/Water =170:25:25:4（by volume）↓

Dry the plate using warm air from a hair dryer.
↓
Spray 0.01% Purimurin/80% acetone solution over the plate.
Dry the plate using warm air from a hair dryer.
↓Mark lipid spots under long-wave UV light (360 nm), using a soft pencil (HB).
The fluorescence image must be taken on a digital camera.
↓Scrape off each spot on medicine paper, and carefully transfer all silica gel
powder into a 15-ml screw-capped glass tube*4.
*4Special attention should be paid not to use any tubes with cracks on themouth or the screw mound.
↓
Add 10 µl each of a 15:0 standard/toluene solution, using a fresh glass capillary
pipette.
↓Add 10 µl of a BHT/toluene solution, using a fresh glass capillary pipette.
(If 14:0 is expected in the lipid samples, BHT must not be added, because
14:0-Me and BHT show the same retention time on a gas chromatograph.)↓Add 3 ml each of HCL/methanol solution. Screw on the tube and mix vigorouslywith a vortex mixer.
↓80 ˚C for 3 hrs on an aluminum heating block.

Day 3Chill all tubes on ice.
↓Extract FA methyl esters with 3 ml distilled hexane.
↓Evaporate hexane, using a vacuum centrifugation evaporator. Dissolve driedlipid residues in ~5 drops of hexane from a Pasteur pipette and subject to gas
chromatograph analyses.
(The hexane solution should be transferred to a 1-ml sample tube for storage ora glass tube for an automatic sample injector.)

Correspondence: Ikuo Nishida, nishida@molbiol.saitama-u.ac.jp