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 100oC for 20 minutes. (Note: each tube should be very well sealed with teflon tape and grease to prevent evaporation.)
- Sonicate and heat at 100oC 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.
