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

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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.