Archived Protocol

<HTML> <HEAD> <META HTTP-EQUIV="Content-Type" CONTENT="text/html; charset=iso-8859-1"> <META NAME="GENERATOR" CONTENT="Mozilla/4.04 [en] (WinNT; U) [Netscape]"> <TITLE>BISC 429</TITLE> </HEAD> <BODY BGCOLOR="#ffffcc"> <div align="center"> <HR> <IMG SRC="title429.gif" ALIGN=CENTER> <HR> </div> <H2> Lipid analysis</H2> <H4>Week 2: Transmethylation of lipids and extraction of methyl derivatives</H4> <A HREF="FFA.gif">Free fatty acids</A> or those which are linked by ester bonds to glycerol backbone, as in <A HREF="lipids.gif">mono-, di-, triglycerides</A> and <A HREF="PL.gif">phospholipids</A>, are usually converted to their methyl esters by transmethylation and analyzed by <A HREF="GLC.html">Gas Liquid Chromatography</A> (week 3). In this laboratory the separated lipids extracted from the silica gel will be transesterified by using BF3 (14% in methanol). Methyl esters can be prepared from all lipid classes using a modified method of <A HREF="references.html">Morrison and Smith (1964)</A>. An advantage of this method is that it is carried out under an atmosphere of N2, so that oxidation of the lipid is minimized.   <H3> EXPERIMENTAL PROTOCOL</H3> 1. Use glass pipettes with bulbs for all pipeting. 2. In the fume hood, pour small amounts of the reagents to be used (except BF3) into clean glass scintillation vials and label properly and leave the remaining in the fume hood. Remove the BF3 bottle from the fridge and let warm up to room temperature. <BLINK>Caution</BLINK>: BF3 is highly corrosive! 3. Set the heating block in the fume hood at 100 °C.   4. Warm the samples extracted from Silica gel to room temp. Flush with N2 for 30 sec.   5. ONLY for the triglyceride extract add <CENTER><TABLE BORDER > <TR> <TD>BF3</TD> <TD>Benzene</TD> <TD>Methanol</TD> </TR> <TR> <TD>350 µl</TD> <TD>300 µl</TD> <TD>350 µl</TD> </TR> </TABLE></CENTER> 6. For the other lipids (PL, MAG, DAG and FFA) add 1 ml of BF3 (open the bottle in the fume hood) i.e. in the ratio of 1 ml of BF3 per 4-16 mg lipid. 7. Close with Green Teflon Coated caps, swirl for a while and place on the heating block for the time periods given below. Make sure that the temperature is 100 °C.   <CENTER><TABLE BORDER > <TR> <TD>Lipid</TD> <TD>time</TD> </TR> <TR> <TD>FFA</TD> <TD>10 min</TD> </TR> <TR> <TD>MAG</TD> <TD>10 min</TD> </TR> <TR> <TD>DAG</TD> <TD>10 min</TD> </TR> <TR> <TD>PL</TD> <TD>10 min</TD> </TR> <TR> <TD>TAG</TD> <TD>45 min</TD> </TR> </TABLE></CENTER>   8. After the given time periods remove from the heating block and leave in the fume hood to cool down to room temperature. 9. Add 1 vol. (1 ml) of distilled H2O (you may use pipetman) and 2 vol. (2 ml) of hexane (again pour into a small clean labeled vial before pipeting; use glass pipette!). 10. Tighten caps, shake well and leave in the fume hood for 1-2 min for phase separation. 11. Using a pasteur pipette, carefully transfer the upper hexane phase into small 2 ml glass vials; this contains fatty acid methyl esters. Be careful not to carry over parts of the aqueous lower phase; it will be difficult to evaporate it. 12. Under a stream of N2, remove the hexane off the sample to complete dryness. 13. Reconstitute in 25 µl of hexane (use a Hamilton syringe to measure). 14. Store at -20 °C until <A HREF="GLC.html">gas chromatography</A> is carried out (week 3). <A HREF="TLC.html"><IMG SRC="arrow_small_leftT.gif" ALIGN=top></A>  <A HREF="GLC.html"><IMG SRC="arrow_small_rightT.gif" ALIGN=RIGHT></A> <HR> [<A HREF="index.html"> BISC 429 home</A>] [<A HREF="phosphatase.html"> Enzyme isolation </A>] [<A HREF="lipoprotein.html"> Lipoprotein isolation </A>] [<A HREF="lipid.html"> Lipid analysis </A>] [<A HREF="DNA.html"> DNA isolation </A>] [<A HREF="mailto:haunerla@sfu.ca"> feedback </A>] </BODY> </HTML>