Archived Protocol

<!doctype html public "-//w3c//dtd html 4.0 transitional//en"> <html> <head> <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1"> <title>R.E.W. Hancock Laboratory: Methods</title> <link rel="StyleSheet" href="bobh.css" type="text/css"> </head> <body> Methylation of Fatty Acids (Kropinski Method) OBJECTIVE: To methylate fatty acids in whole cells or lipopolysaccharide. REAGENTS : <ul> <li>Methanol-Hydrochloride Reagent Kit <li>>10mg of whole cells or 1 mg of lipopolysaccharide <li>400 nmoles of fatty acid standard (pentadecanoic acids C15) </ul> METHODS: <ol> <li>Prepare 1M MeOH-HCl reagent according to the instruction given with the kit. <li>Add internal standard (C15 fatty acid) to give a final concentration of 400 nmoles/100ml (usually 10mg of C15 in 100ml of reagent. <li>Weigh 10mg of whole cells or 1 mg of LPS into a clean screw-cap tube. <li>Add 1ml of MeOH-HCl reagent/internal standard into each tube and vortex. <li>Heat at 100oC for 20 minutes. (Note: each tube should be very well sealed with teflon tape and grease to prevent evaporation.) <li>Sonicate and heat at 100oC overnight. <li>Neutralize acidity with 0.5N NaOH. Test pH with pH paper. <li>Centrifuge in clinical centrifuge for 5 minutes. Save supernatent in clean glass vial. <li>Do gas chomatography with programmed REWH method. </ol> </body> </html>