I'm trying to separate small metabolites from yeast lysate prior to FMOC-derivatization and HPLC analysis. The cleaner I can get the lysate the better but I'm on the fence when it comes to buying $$$-products-$$$ for sample prep.
Do you know of a simple method to separate small metabolites from larger macromolecules (DNA, proteins, etc.)?
Could I add chloroform to the cell lysate, thus removing soluble proteins and lipids from aqueous phase?
TCA precipitation works well to remove most of the macromolecules. Protocols available in Methods in Enzymology.
Try Folch biphasic extraction method. This is general for metabolomic techniques. Combine MeOH and chloroform phases and evaporate to dryness. Dissolve the residue using the appropriate solvent for FMOC derivatization...Solvent types and volumes may be modified depending on the particular small molecule groups. Other than chemical approaches, you may use UF in the format of S-TRAP, FASP for small volumes, and stirred cells for large volumes as physical isolation alternatives.
Good luck
Run the (cleared) lysate through a desalting column (PD10, NAP10, etc. or make your own with Sephadex G10 or G25) and keep the low molecular weight (salt) fraction(s).
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