Please read Metabolite Processing Precautions
before proceeding.
⦁ Prepare and separate all material and solutions before you start. Check the Recommended Resources List.
⦁ Prepare two tubes labeled as QC (pooled quality control). NOTE: One tube will be for the polar (QCp) fraction
nd the other for the non-polar fraction (QCnp). If only one LC method is used, you may combine the fractions thus using only one QC tube.
⦁ Allow the cells thaw in ice.
⦁ Add 1 mL of ice-cold extraction solution F to the cells pellets.
⦁ Vortex until pellet dissolves.
⦁ Transfer each sample to a new pre-cooled 2 mL tube containing 80 mg of zirconium beads.
⦁ Homogenize cells by beating for 10 s and then cooling down on ice for 30 s. Repeat the cycle 3 more times.
⦁ Centrifuge the solution at 10,500 g for 10 min at 4 °C.
⦁ Collect the supernatant into a new 2 mL labeled tube (polar fraction).
⦁ Keep the tubes in dry ice and proceed to the next step.
⦁ IMMEDIATELY after collection of the polar fraction, add 1 mL of ice-cold extraction solution E (Non-polar extraction).
⦁ Repeat the homogenization step.
⦁ Centrifuge the mixture for 10 min at 15000 g and 4°C.
⦁ From the bi-phasic system created, transfer the upper layer to the tube containing the polar fraction.
⦁ Carefully aspirate the bottom organic layer and transfer into another 2 mL Eppendorf tube.
⦁ From the polar fractions, take 10% of each sample and transfer to the QCp labeled tube. Do the same
procedure for the non-polar fraction (QCnp tube).
⦁ Freeze-dry the polar fractions in a lypholyzer and the non-polar extracts in a glass cupboard (lypholyzers ARE NOT compatible with chloroform).
⦁ PAUSE POINT Store the samples at -80°C for up to 12 weeks.