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Extraction of glycolipids
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Extraction of glycolipids

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Introduction Protocol References Credit lines
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

chloroform (CHCl3)

methanol (MeOH)

CHCl3-MeOH mixture (C/M, volume/volume)

4 M NaOH

4 M acetic acid

distilled water (DW)

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.)

sonic bath

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.

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3) 

 Add 1.2 mL of ice-cold MeOH and mix well.

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4) 

 Add 2 mL of CHCl3 and mix well.

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5) 

 Incubate at 37°C for 1 h with shaking.

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6) 

 Add 1 mL of MeOH and mix well.

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7) 

 Centrifuge at 1,000 × g for 10 min.

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8) 

 Transfer the supernatant to a glass vial.

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9) 

 Add 2 mL of CHCl3/MeOH/DW (1/2/0.8, v/v/v) to the pellet obtained at 7).

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10) 

 Incubate at 37°C for 2 h with shaking.

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11) 

 Centrifuge at 1,000 × g for 10 min.

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12) 

 Withdraw the supernatant and transfer to the pooled extracts obtained at 8).

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13) 

 Dry with a Speed Vac concentrator.

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2.

Extraction of GSLs from cultured cells

1) 

 Harvest the cells (~1 × 106 cells).

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2) 

 Add 2 mL of CHCl3/MeOH (2/1, v/v).

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3) 

 Sonicate in a sonic bath for 5 min.

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4) 

 Incubate at 37°C for 1 h with shaking.

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5) 

 Centrifuge at 1,000 × g for 10 min.

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6) 

 Withdraw the supernatant and transfer to a glass vial.

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7) 

 Add 1 mL of CHCl3/MeOH/DW (1/2/0.8, v/v/v) to the pellet obtained at 5).

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8) 

 Incubate at 37°C for 2 h with shaking.

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9) 

 Centrifuge at 1,000 × g for 10 min.

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10) 

 Withdraw the supernatant and transfer to the pooled extracts obtained at 6).

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11) 

 Dry with a Speed Vac concentrator.

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3.

Saponification and desalting

1) 

 Dissolve the sample in 2 mL of MeOH.

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2) 

 Add 25 μL of 4 M NaOH.

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3) 

 Incubate at 37°C for 2 h.

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4) 

 Add 25 μL of 4 M acetic acid.

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5) 

 Add 2 mL of water.

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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).

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8) 

 Wash the cartridge with 1 mL of DW.

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9) 

 Elute the GSLs with 1 mL of MeOH.

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10) 

 Dry with a Speed Vac concentrator.

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4.

Separation of neutral and acidic GSLs

1) 

 Dissolve the sample in 2 mL of MeOH.

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2) 

 Add 2 mL of water.

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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).

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5) 

 Wash with 1 mL of DW.

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6) 

 Elute the neutral GSLs with 1 mL of MeOH.

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7) 

 Dry with a Speed Vac concentrator (neutral GSL fraction).

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8) 

 Elute the acidic GSLs with 1 mL of MeOH/0.2 M ammonium acetate (acidic GSL fraction).

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9) 

 Add 1 mL of DW to 1 mL of the acidic GSL fraction.

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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

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