Hancock Lab

Methylation of Fatty Acids (Kropinski Method)

Categorized: Outer Membranes

Objective:

To methylate fatty acids in whole cells or lipopolysaccharide.

Reagents:

  • Methanol-Hydrochloride Reagent Kit
  • 10mg of whole cells or 1 mg of lipopolysaccharide
  • 400 nmoles of fatty acid standard (pentadecanoic acids C15)

Methods:

  1. Prepare 1M MeOH-HCl reagent according to the instruction given with the kit.
  2. Add internal standard (C15 fatty acid) to give a final concentration of 400 nmoles/100ml (usually 10mg of C15 in 100ml of reagent.
  3. Weigh 10mg of whole cells or 1 mg of LPS into a clean screw-cap tube.
  4. Add 1ml of MeOH-HCl reagent/internal standard into each tube and vortex.
  5. Heat at 100oC for 20 minutes. (Note: each tube should be very well sealed with teflon tape and grease to prevent evaporation.)
  6. Sonicate and heat at 100oC overnight.
  7. Neutralize acidity with 0.5N NaOH. Test pH with pH paper.
  8. Centrifuge in clinical centrifuge for 5 minutes. Save supernatent in clean glass vial.
  9. Do gas chomatography with programmed REWH method.

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