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
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.
