Metabolite extraction from supernatant cells

Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom

Methanol/Chloroform extraction of whole cell small molecular weight metabolites from cells in suspension.

• Pre-chill methanol on dry-ice
• Transfer cell culture to 15ml tube to wash.
• Centrifuge at 1500rpm for 5 minutes to pellet. Add 5ml pre-warmed PBS, resuspend and repeat x2
• Centrifuge at 1500rpm for 5 minutes to pellet. Resuspend in 1ml pre-warmed PBS.
• Transfer cells to champagne flute glass vials. Centrifuge at 1200rpm for 5 minutes to pellet.
• Pre-chill centrifuge to 4’C.

Pre-chill chloroform and dH20 on wet ice.

• Completely remove supernatant PBS, then resuspend pellet in 400ul pre-chilled methanol. Place glass vials on dry ice to freeze rapidly.

OPTIONAL: Samples may now be stored at -80’C until required.

• Pipette 325ul dH20 via pipette onto each sample.
• Syringe 400ul chloroform via Hamilton syringe onto each sample (chloroform will digest plastic).
• Keep samples at 4’C for 10 minutes to allow phases to separate.
• Centrifuge at 1200rpm for 10 minutes at 4’C in swingout rotor.
• Transfer vials to the bench at room temperature for 5 minutes.
• Remove ~400ul upper (polar) layer into eppendorf using by pipette.
• Remove ~200ul lower (non-polar) layer into glass vials using Hamilton syringe.
• To dry samples for re-suspension in analysis buffer, place samples in vacuum dryer. Polar fraction will dry within 3-4 hours.