Phosphate Assay by Suprya Jaydev

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

Procedure1) Prepare standards in duplicate.--> standards: 0, 3, 5, 7, 10, 12, and 15 µl of a 1 mM NaH2PO4 stock solution2) 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 molybdate0.1 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.