Phosphate Assay – Lipids

Protocol

Introduction: This can be used for determining the phospholipids content. Be careful when

adding acid to the tubes. Most work should be done in the hood. It is best to practice using old

samples and a standard curve before using important experimental samples.

Protocol: Clin Chem Acta 121, 111-116. 1982

1 - Prepare a standard curve phosphate curve in triplicate.

(0, 25, 50, 75, 100, 150, 200, 400 μl of 1 mM KH2PO4) – calculate how many μg of phosphate are in each

tube you will need this later.

2 - Add 25, μl of sample in separate tubes. Do in triplicate.

3 - Dry standards in heating block inside hood

- dry organic solvent (lipid samples) under nitrogen.

4 - Add 100 μl concentrated H2SO4 to all tubes.

5 - Vortex and put tubes in the heating block for 10 min.

6 - Allow tubes to cool to room temperature and add 50 μl of 6% hydrogen peroxide

7 - Vortex tubes and place in heating block for 40 minutes.

8 - Allow tubes to cool and add 2 ml of H2O, mix well.

9 - Add 800 μl of Color reagent to each tube.

10 - Boil samples on hot plate or heat block for 10 - 15 minutes or until highest standard turns a very dark blue.

11 - Transfer to 1 ml cuvettes and read absorbance at 797 nm

12 - Plot standards as absorbance vs. μg phosphate

Solutions

50 1 mM KH2PO4

Color reagent

1:1 ammonium anhydride molybdic acid (0.625g/50ml): ascorbic acid (0.45 g / 50 ml)

Mix just prior to use.

6% H2O2 - (1 ml of 30% H2O2 and 4 ml of H2O) Make just prior to use

http://www.mnstate.edu/provost/PhosphateAssayprotocol.pdf

Wallert and Provost Lab