Archived Protocol

<HTML> <HEAD> <TITLE>Phosphate Assay by Suprya Jaydev</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>Phosphate Assay by Suprya Jaydev</CENTER> </H2> Contributor: Suprya Jayadev Date: Sep. 8, 1994 Reagents Ashing buffer: 10 g Mg(NO3)2 100 ml EtOH 1.5 N HCl stock: 119.7 ml concentrated HCl (11.6M @ 36% by weight) 880.3 ml H2O 1 N Sulfuric acid stock: 28.9 ml concentrated sulfuric acid (18M/36N @ 96% by weight) 971.1 ml H2O Ammonium molybdate stock: 4.2 ml Ammonium molybdate 1000 ml 1N H2SO4 <HR> Procedure 1) Prepare standards in duplicate. --> standards: 0, 3, 5, 7, 10, 12, and 15 µl of a 1 mM NaH2PO4 stock solution 2) Aliquot lipid unknowns. 3) Add 100 µl ashing buffer to each sample. 4) Dry EtOH and chloroform from samples by heating @ 80°C for ~20 minutes. 5) Flame samples using a strong flame on the bunsen burner. 6) Remove samples from flame when the brown gas ceases to be released. --> There should be a white percipitate remaining at the base of the tubes. If the ashing process is allowed to proceed for too long, the percipitate will appear brown/charred and this will skew the spectrophotometer readings. 7) Add 0.3 ml of 0.5N HCl to each sample and boil for 15 minutes. 8) To each tube, add: 0.6ml ammonium molybdate 0.1 ml ascorbic acid (10% w/v, made fresh EACH time) VORTEX!! 9) Incubate for 30 minutes @ 45°C or for 60 minutes @ 37°C. 10) Read OD820. </BODY> </HTML>