TG extraction from seeds
Day 1
Lipid extraction by Folch’s method; solvent evaporation; weighing; storage at-30˚C.
Day 2
Separation of lipids by TLC; recovery of lipid spots; antioxidant addition;
HCl/methanol treatment for GLC.
Days 3 and 4
Hexane extraction; GLC analysis.

Lipid extraction from Arabidopsis seeds
Sampling Table
Sample name (date):Sample No.
Fresh weight (mg)
H2O from material(ml) ––– (W)（生重量 x0.9）
2-Propanol(ml)Homogenate (ml)Methanol
Chloroform (1)Pellet washing (ml)Methanol
Chloroform (2)Example

No. 1

No. 2

No. 3

IgnoreIgnoreIgnoreIgnore

111

1

0

0

0

0

2

222

1

1

112222

６６

６６

1.2

1.2

1.2

1.2

10Combined
supernatant(A + B in the text)
0.9% KCl

Empty vessel (g)Total lipid extracts(mg)Day 1
Place 1 ml 2 propanol in a 10 ml screw-capped glass tube and keep the tube ina 90 ˚C water bath.
↓Add weighed seeds (10 mg) to the tube and keep for another 5 min.
↓Chill the tube on ice.
↓Add 2 ml chloroform, and homogenize the seeds, using a Polytron (max.
speed).
↓3000 rpm x 15 min (r.t.)
Recover the supernatant (A) into a fresh 10-ml screw-capped glass tube bydecantation (if the pellet is soft, remove by pipetting).
↓Resuspend the pellet in 1 ml methanol and 2 ml chloroform (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 1.2 ml (0.2 vol) of 0.9% KCl and mix well by vigorous shaking.
↓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 dryweight.
↓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 BHT should be added at a final
concentration of 10 µg/ml.

Day 2
Separation of lipids by silica gel thin-layer chromatography
Using a 100-µl glass micro syringe, streak 100 µl of the lipid extract (1 mg lipidequivalent) on a 1-cm line marked 2 cm from the bottom of TLC plate (20 x 20cm, Silica gel 60, Merck 1.05721.) as shown below.
When streaking lipids

1st1st dimension：
Hexane/diethyl ether/acetic acid = 80:30:1（by volume)↓Air-dry the plate (you may use 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 capillarypipette.
↓
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 h 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