Hancock Lab

Phosphate Assay

Categorized: Biochemical Assays

Stock Solution:

0.001 M Phosphate Solution

use 100, 200, 300, 400, 500 µl and make up each sample to 500 µl final volume.

Reagents:

  1. 10% Magnesium Nitrate in ethanol.
  2. 1 M HCl.
  3. 10% Ascorbic Acid (1 gm/10 ml).
  4. Ammonium Molybdate, .42% in 1 N Sulphuric Acid

(.42 gm + 2.66 ml c/H2SO4 made up into 100 ml)

Method:

  1. Weigh 0.5 mg LPS into Pyrex tube.
  2. Add 0.05 ml 10% Magnesium Nitrate in ethanol.
  3. Evaporate to dryness on flame.
  4. Cool. Add 0.3 ml 1 N HCl. Heat at 100oC for 15 min. in a boiling water bath.
  5. Add 0.7 ml Ascorbic/Molybdate (1 part to 6 parts freshly mixed).
  6. Keep at 45oC for 20 min.
  7. Read absorbance at 820 nm against reagent blank. (absorbence of 0.240 for 0.01 µmol phosphate).

* Ascorbic Acid is stable at 2 – 4oC for approx. 7 weeks.

Method Categories