MethylationofFattyAcids(KropinskiMethod)OBJECTIVE:Tomethylatefattyacidsinwholecellsorlipopolysaccharide.REAGENTS:

• Methanol-HydrochlorideReagentKit
• >10mgofwholecellsor1mgoflipopolysaccharide
• 400nmolesoffattyacidstandard(pentadecanoicacidsC15)

1. Prepare1MMeOH-HClreagentaccordingtotheinstructiongivenwiththekit.
2. Addinternalstandard(C15fattyacid)togiveafinalconcentrationof400nmoles/100ml(usually10mgofC15in100mlofreagent.
3. Weigh10mgofwholecellsor1mgofLPSintoacleanscrew-captube.
4. Add1mlofMeOH-HClreagent/internalstandardintoeachtubeandvortex.
5. Heatat100^oCfor20minutes.(Note:eachtubeshouldbeverywellsealedwithteflontapeandgreasetopreventevaporation.)
6. Sonicateandheatat100^oCovernight.
7. Neutralizeaciditywith0.5NNaOH.TestpHwithpHpaper.
8. Centrifugeinclinicalcentrifugefor5minutes.Savesupernatentincleanglassvial.
9. DogaschomatographywithprogrammedREWHmethod.

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