MethylationofFattyAcids(KropinskiMethod)

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:

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

6. Sonicate and heat at 100^oC 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.