Total lipids are extracted from samples with a mixture of chloroform, methanol and water. To remove the glycerolipids, saponification is carried out with NaOH. Neutral glycosphingolipids (GSLs) are separated from acidic GSLs by anion exchange chromatography. |
Category | Glycolipids and related compounds |
Protocol Name | Extraction of glycolipids |
Authors
|
Okino, Nozomu
*
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
Ito, Makoto
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
|
KeyWords |
|
Reagents
|
● |
|
● |
|
● |
CHCl3-MeOH mixture (C/M, volume/volume) |
● |
|
● |
|
● |
|
|
Instruments
|
● |
OASIS HLB cartridge 1 cc/30 mg (reverse-phase cartridge, Waters Corp., Milford, MA) |
● |
OASIS MAX cartridge 1 cc/30 mg (anion exchange cartridge, Waters Corp.) |
● |
|
● |
Speed Vac concentrator (Thermo Fisher Scientific Inc., Waltham, MA) |
|
Methods |
1. |
Extraction of GSLs from tissues
|
1) |
Homogenize the sample with 4 vol of ice-cold water (approximately 0.25 g/ mL). |
Comment 1
|
|
2) |
Transfer 200 μL of sample (~50 mg of tissue) to a glass vial. |
Comment 0
|
|
3) |
Add 1.2 mL of ice-cold MeOH and mix well. |
Comment 0
|
|
5) |
Incubate at 37°C for 1 h with shaking. |
Comment 0
|
|
7) |
Centrifuge at 1,000 × g for 10 min. |
Comment 0
|
|
8) |
Transfer the supernatant to a glass vial. |
Comment 0
|
|
9) |
Add 2 mL of CHCl3/MeOH/DW (1/2/0.8, v/v/v) to the pellet obtained at 7). |
Comment 0
|
|
10) |
Incubate at 37°C for 2 h with shaking. |
Comment 0
|
|
11) |
Centrifuge at 1,000 × g for 10 min. |
Comment 0
|
|
12) |
Withdraw the supernatant and transfer to the pooled extracts obtained at 8). |
Comment 0
|
|
13) |
Dry with a Speed Vac concentrator. |
Comment 0
|
|
|
2. |
Extraction of GSLs from cultured cells
|
1) |
Harvest the cells (~1 × 106 cells). |
Comment 0
|
|
2) |
Add 2 mL of CHCl3/MeOH (2/1, v/v). |
Comment 0
|
|
3) |
Sonicate in a sonic bath for 5 min. |
Comment 0
|
|
4) |
Incubate at 37°C for 1 h with shaking. |
Comment 0
|
|
5) |
Centrifuge at 1,000 × g for 10 min. |
Comment 0
|
|
6) |
Withdraw the supernatant and transfer to a glass vial. |
Comment 0
|
|
7) |
Add 1 mL of CHCl3/MeOH/DW (1/2/0.8, v/v/v) to the pellet obtained at 5). |
Comment 0
|
|
8) |
Incubate at 37°C for 2 h with shaking. |
Comment 0
|
|
9) |
Centrifuge at 1,000 × g for 10 min. |
Comment 0
|
|
10) |
Withdraw the supernatant and transfer to the pooled extracts obtained at 6). |
Comment 0
|
|
11) |
Dry with a Speed Vac concentrator. |
Comment 0
|
|
|
3. |
Saponification and desalting
|
1) |
Dissolve the sample in 2 mL of MeOH. |
Comment 0
|
|
6) |
Apply the sample to a OASIS HLB cartridge. |
Comment 1
|
|
7) |
Wash the cartridge with 1 mL of MeOH/DW (1/1, v/v). |
Comment 0
|
|
8) |
Wash the cartridge with 1 mL of DW. |
Comment 0
|
|
9) |
Elute the GSLs with 1 mL of MeOH. |
Comment 0
|
|
10) |
Dry with a Speed Vac concentrator. |
Comment 0
|
|
|
4. |
Separation of neutral and acidic GSLs
|
1) |
Dissolve the sample in 2 mL of MeOH. |
Comment 0
|
|
3) |
Apply the sample to an OASIS MAX cartridge (anion exchange cartridge). |
Comment 1
|
|
4) |
Wash with 1 mL of MeOH/DW (1/1, v/v). |
Comment 0
|
|
6) |
Elute the neutral GSLs with 1 mL of MeOH. |
Comment 0
|
|
7) |
Dry with a Speed Vac concentrator (neutral GSL fraction). |
Comment 0
|
|
8) |
Elute the acidic GSLs with 1 mL of MeOH/0.2 M ammonium acetate (acidic GSL fraction). |
Comment 0
|
|
9) |
Add 1 mL of DW to 1 mL of the acidic GSL fraction. |
Comment 0
|
|
10) |
Apply the acidic GSL fraction to an OASIS HLB cartridge (normal-phase cartridge). |
Comment 1
|
|
11) |
Wash the cartridge with 1 mL of MeOH/DW (1/1, v/v). |
Comment 0
|
|
12) |
Wash the cartridge with 1 mL of DW. |
Comment 0
|
|
13) |
Elute the acidic GSLs with 1 mL of MeOH. |
Comment 0
|
|
14) |
Dry with a Speed Vac concentrator (acidic GSL fraction). |
Comment 0
|
|
|
Figure & Legends |
Figure & Legends
Fig.1. Scheme for the extraction and separation of GSLs from biological samples |
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
|
Date of registration:2014-07-31 10:02:19 |
- Waki, H., Kon, K., Tanaka, Y., and Ando, S. (1994) Facile methods for isolation and determination of gangliosides in a small scale: age-related changes of gangliosides in mouse brain synaptic plasma membranes. Anal Biochem. 222, 156–162 [PMID : 7856842]
- Oshima T et al. (2000) Kisoseikagakujikkenhou vol. 5 Shishitsu/Toushitsu/Fukugoutoushitsu, Tokyo kagakudoujin (in Japanese).
|
This work is licensed under Creative Commons Attribution-Non-Commercial Share Alike. Please include the following citation
How to Cite this Work in an article:
Okino, Nozomu,
Ito, Makoto,
(2014). GlycoPOD https://jcggdb.jp/GlycoPOD.
Web.28,3,2024 .
How to Cite this Work in Website:
Okino, Nozomu,
Ito, Makoto,
(2014).
Extraction of glycolipids.
Retrieved 28,3,2024 ,
from https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t4.
html source
Okino, Nozomu,
Ito, Makoto,
(2014).
<b>Extraction of glycolipids</b>.
Retrieved 3 28,2024 ,
from <a href="https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t4" target="_blank">https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t4</a>.
Including references that appeared in the References tab in your work is
much appreciated.
For those who wish to reuse the figures/tables, please contact JCGGDB
management office (jcggdb-ml@aist.go.jp).
|
|