<HTML> <HEAD> <TITLE>Purification of Demethylated Sphingomyelin</TITLE> </HEAD> <BODY> <P><CENTER>[ <A HREF=http://www.musc.edu/bcmb/ceramide/protocols/toc.html>TOC</A> | <A HREF=http://www.musc.edu/bcmb/ceramide/protocols/apoptosis.html>Apoptosis</A> | <A HREF=http://www.musc.edu/bcmb/ceramide/protocols/biochemistry.html>Biochemistry</A> | <A HREF=http://www.musc.edu/bcmb/ceramide/protocols/cbcc.html>CBCC</A> | <A HREF=http://www.musc.edu/bcmb/ceramide/protocols/lipid.html>Lipid</A> | <A HREF=http://www.musc.edu/bcmb/ceramide/protocols/mbg.html>MBG</A> | <A HREF=http://www.musc.edu/bcmb/ceramide/protocols/yeast.html>Yeast</A> | <A HREF=http://www.musc.edu/bcmb/ceramide/protocols/misc.html>Misc</A> ]<BR> <HR></CENTER> <H2><CENTER>Purification of Demethylated Sphingomyelin</CENTER> </H2> <BR> <BR> Contributor: Suprya Jayadev<BR> Date: Feb. 20, 1991<BR> <BR> <BR> <BR> 1) Pack a Bio-Sil A column as follows:<BR> <BR> a) Pack base of column with a small portion of glass wool.<BR> <BR> b) Suspend 20 grams of 100-200 mesh BioSil A in 100 ml chloroform.<BR> <BR> c) Pour silica/chloroform mixture into column eluting chloroform from base.<BR> <BR> - Press out any air bubbles in the glass wool as the gel is initially being poured. While pouring column matrix, stir continuously with a stir bar in order to prevent bubble formation in the column.<BR> <BR> d) Wash column through with "200 ml chloroform.<BR> <BR> - ALWAYS maintain a fluid level above the packing silica... NEVER let column dry out!!<BR> <BR> 2) Dry pooled lower phases from the demethylation reaction using the rotovapor system.<BR> <BR> --> DO NOT heat!<BR> <BR> 3) Resuspend dried lipid in a minimal volume of chloroform and carefully layer over packed column.<BR> <BR> 4) Allow sample to run into column while maintaining a small level of chloroform above the packed silica.<BR> <BR> 5) Sequentially elute with the following solvents:<BR> <BR> a) 100 ml chloroform<BR> b) 200 ml chloroform/methanol (20:1)<BR> c) 200 ml chloroform/methanol (9:1)<BR> d) 200 ml chloroform/methanol (5:1)<BR> e) 200 ml chloroform/methanol (4:1)<BR> f) <B>800 ml</B> chloroform/methanol (3:1)<BR> g) 200 ml chloroform/methanol (3:1)<BR> <BR> 6) Dry down fractions (f) & (g) separately and re-suspend in 4 ml of chloroform/methanol (1:1).<BR> <BR> 7) Spot 2 µl of each fraction and a small portion of DMSM & SM standard onto a TLC plate.<BR> <BR> 8) Run TLC in chloroform/methanol/ammonium hydroxide (60:35:8) and develop spots using iodine vapor then potassium permanganate spray.<BR> <BR> --> Fraction (f) should contain the DMSM.<BR> <BR> 9) Completely dry down the DMSM containing fraction.<BR> <BR> 10) Determine gram weight of DMSM recovered.<BR> <BR> 11) Resuspend in 2 ml of methanol and store at -20°C until needed for labeled SM synthesis.<BR> <BR> <BR> </BODY> </HTML>