Methylation of Fatty Acids (Kropinski Method)

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