Homogeneous and simple procedure. Simple “mix-and-measure” procedure allows reliable quantitation of ALP activity within 5 minutes.

Robust and amenable to HTS. All reagents are compatible with highthroughput liquid handling instruments.

Characterization and Quality Control for ALP production.

Drug Discovery: high-throughput screen for ALP inhibitors and evaluation of ALP inhibitors.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

PROCEDURES. This assay is based on a kinetic reaction. Use of a multi-channel pipettor is recommended. Addition of Working Reagent to samples should be quick and mixing should be brief but thorough. Assays can be executed at room temperature or 37°C.

Reagent preparation: equilibrate reagents to room temperature. The Working Solution is prepared by mixing for each 96-well assay, 200 μL Assay Buffer, 5 μL Mg Acetate (final 5 mM) and 2 μL pNPP liquid substrate (10 mM). Fresh reconstitution is recommended, although the Working Solution is stable for at least one day at room temperature.

Sample preparation: ALP is stable for 48 hours at 4°C and 2 months at - 20°C. EDTA, oxalate, fluoride, citrate are known inhibitors of ALP and should be avoided in sample preparation. Serum, plasma (no EDTA/citrate, ideally unhemolyzed) and cell culture media can be assayed directly. To measure intracellular ALP, cell lysate can be prepared as follows: 104 cells are washed with PBS and lysed in 0.5 mL 0.2% Triton X-100 in distilled water by shaking for 20 min at room temperature.

1. Transfer 200 μL distilled water (H2O) and 200 μL Calibrator into separate wells of a clear bottom 96-well plate.

2. Carefully transfer 5 to 50 μL samples into other wells.

3. Pipet 150 to 195 μL Working Solution to sample wells. The final reaction

volume in the sample wells should be 200 μL. Tap plate briefly to mix.

4. Read OD405nm (t = 0), and again after 4 min (t = 4 min) on a plate reader.

5. Calculation: ALP activity of the sample (IU/L = μmol/(L·min)) is

1. Transfer 50 μL samples into 1-cm cuvettes.

2. Pipet 950 μL Working Solution to samples. Mix briefly.

3. Read OD405nm shortly after the mixing, and again after 4 min.

4. Calculation: ALP activity of the sample (IU/L) is

Note: (1) if sample ALP activity exceeds 800 IU/L, dilute samples in saline and repeat the assay, multiply the result by the dilution factor. (2) incubation can be prolonged for samples with low ALP activity.

Pipeting devices and accessories (e.g. multi-channel pipettor).

Clear bottom 96-well plates (e.g. Corning Costar) and plate reader.

Spectrophotometer and cuvets for measuring OD 405nm.

EXAMPLES. Samples were assayed in duplicate (n = 2) using the 96-well plate protocol. The ALP activity (U/L) was 13.4 ± 0.4 for a human serum, 190.4 ± 1.6 for rat serum and 202.8 ± 4.3 for goat serum.

with increasing ALP concentration

1. Kim, H.J. et al (2006) Glucocorticoids suppress bone formation via the osteoclast. J. Clin. Invest. 116:2152–2160.

2. Bhattacharya, A. et al (2006). Effect of fish oil on bone mineral density in aging C57BL/6 female mice. J. Nutr. Biochem 18(6):372-379.

3. Wan, Y., Chong, L-W., & Evans, R.M. (2007). PPAR-g regulates osteoclastogenesis in mice. Nature Med. 13(12): 1496-1503.