We use an ELISA test system that work quite well to measure the activity of the ALP

Measurement of Alkaline Phosphatase Activity (TNAP)

Reagents:

Alkaline Phosphatase lysis buffer:

Tris: 1.5M (pH 10)

ZnCl2: 1mM

MgCl2: 1mM

Triton X-100: 1%

Phenylmethylsulfonylfluoride (PMSF): 100 mM

Aprotinin

Alkaline Phosphatase sample buffer:

Diethanolamine (DEA): 0.1M (pH 9.8)

Triton X-100: 0.1%

para-nitrophenyl phosphate (pNPP)

sodium hydroxide (NaOH): 100 mM

Preparation of solutions:

Prepare all solutions (see reagents) and store them in labeled glass bottles at 4°C.

AP lysis buffer:

90.85 g Tris (adjust pH to 10 with HCl pH)

500 µl 1M ZnCl2 (0.136 g to 1 ml H2O)

500 µl 1M MgCl2 (0.203 g to 1 ml H2O)

5 ml Triton X-100

Fill up to 500 ml with H2O

add 1:1000 aprotinin and 1:100 PMSF each time fresh before use

PMSF (100 mM) Dissolve 0.0174 g in 1 ml isopropanol (Final concentration in lysis buffer 1 mM)

AP sample buffer:

5.25 g DEA (adjust to pH 9.8)

500 µl Triton X-100

Fill up to 500 ml with H2O

para-nitrophenyl phosphate (pNPP):

13.5 mg to 10 ml AP sample buffer

NaOH (100 mM)

Carrying out the cell lysis:

- Wash cells 3x with PBS (300 µl)

- Incubate 100 µl AP lysis buffer/24-well 30 min

- Scrape off cells with pipette and transfer solution to Eppi

- 3 x 30s vortexing

- Freezing (-20°C) or further processing of samples

- Centrifugation: 30 min, 4°C, 13.000 rpm

- Continue to use supernatant

Performing the activity measurement:

- 10 µl lysate in 96-well plate (3-fold determination)

- Add 100 µl AP sample buffer with pNPP: 30 min, 37°C

- Add 100 µl NAOH (reaction stop)

- Measure extinction at 405nm (reference: 655nm)

To determine the activity of the alkaline phosphatase it is advisable to generate a calibration curve.