Archived Protocol

<HTML> <HEAD> <TITLE>Preparation of bOG-DOPG mixed micelles</TITLE> </HEAD> <BODY> <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> ] <HR></CENTER> <H2><CENTER>Preparation of bOG-DOPG mixed micelles</CENTER> </H2> Contributor: Date: November 22, 1993 <H3>Materials:</H3> <BLOCKQUOTE>All glassware must be acid washed, rinsed thoroughly with water then rinsed with acetone and dried.</BLOCKQUOTE> <BLOCKQUOTE>Redistill acetone.</BLOCKQUOTE> <HR> Recrystallizing BOG: 1) Add 5 grams of Octyl-b-D-glucopyranoside (bOG = Calbiochem #494459) to 20 ml acetone. 2) Heat mixture at 40°C to completely dissolve the bOG. 3) Filter solution through a Pyrex® scintered glass funnel. 4) Slowly add 100 ml diethyl ether to the filtered solution and re-heat if necessary (if precipitation appears in the solution). 5) Place in -20°C freezer overnight (or longer) to allow crystals to form. 6) Pour solution containing recrystallized bOG through a scintered glass funnel. 7) Wash crystals with 500 ml ice cold diethyl ether. 8) Completely dry crystals and scrape out of funnel. 9) Weigh crystals and store as powder in the -20 °C freezer. <HR> Preparing mixed micelles: 10) Aliquot 0.97 ml of 20 mg/ml L-a-dioleoyl-phosphatidylglycerol (DOPG sodium salt = Avanti #840475) into Pyrex® screw-cap tubes. --> NOTE: DOPG stock solution should be 20 mg/ml or 27 mM in chloroform. 11) Dry the DOPG as a thin film around the bottom of the glass tube. --> NOTE: If the DOPG is dried as a pellet at the bottom of the tube it is more difficult (takes more time) to dissolve in aqueous solution. 12) Make up a 7.5% solution of bOG in water and add 1 ml to each tube containing dried DOPG. 13) Sonicate mixture until the DOPG is completely dissolved. --> Do 4 minute bursts with 5 minute rests in between. Depending on how the DOPG was dried in the tube, dissolving the DOPG completely will take 2 to 4 hours. </BODY> </HTML>