Polar lipids
Polar lipids were extracted by using petroleum
ether as described by Minikin et al., 1984. The steps are as follows:
- Add 2 mL of methanol:watera
(100:10) to a glass tube having 50 mg of freeze dried cell.
- heat
the tube to 60-80°C and add 2mLof petroleum ether, capped tightly.
- Rotate
the tube for15 min and remove upper layer.
- Add
1 mL of petroleum ether, mix again for 15min and remove upper layer.
- Heat
the lower layer in boiling water bath for 5min, cool in water bath at 37°C for
5 min.
- Add
2.3 mL of chloroform-methanol-water (90:100:30).
- Mix
for 60 min, centrifuge and transfer supernatant to the new tube.
- Add
0.75 mL of chloroform-methanol-water (50:100:40).
- Mix
for 30 min, centrifuge and added to supernatant combined to above.
- Add
0.75 mL of chloroform-methanol-water (50:100:40) and treat as above step.
- Add
1.3 mL of chloroform and 1.3 mL of watera to combined supernatants.
- Mix
thoroughly, centrifuge, remove upper layer, and evaporate the lower layer using
rotary vacuum evaporator.
*a: 0.3%
aqueous NaCl.
The extracted elution
of polar lipid was dissolved in 200 µL of chloroform-methanol (2:1, v/v). The polar lipids were analysed by two-dimensional thin layer
chromatography (TLC) using chloroform/methanol/water (65:25:4; v/v/v) in the
first dimension and chloroform/methanol/acetic acid/water (40:7.5:6:2, v/v/v/v)
in the second. Appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987) were used to identify
the spots; molybdophosphoric acid (phosphomolybdic acid reagent, 5% v/v
solution in ethanol; Sigma-Aldrich, Germany) was used to detect total polar
lipids, ninhydrin reagent (0.2% solution; Sigma Life Science, USA) was used to
detect amino lipids, Zinzadze reagent (molybdenum blue spray reagent, 1.3%;
Sigma Life Sciences) was used to detect phospholipids, and α-naphthol reagent
was used to detect glycolipids.
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