• Purity

  >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave a fluorogenic substrate, 4-Methylumbelliferyl phosphate (4-MUP). The specific activity is >30,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Leu18-Ser502, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Leu18

• Predicted Molecular Mass

  54 kDa

• SDS-PAGE

  65-75 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 1 mM MgCl₂, pH 9.0

• Recombinant Human Alkaline Phosphatase/ALPL/TNAP (rhTNAP) (Catalog # 2909-AP)

• Substrate: 4-Methumbelliferly phosphate (4-MUP) (Calbiochem, Catalog # 474431), 10 mM in deionized water

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhTNAP to 0.01 ng/µL in Assay Buffer.

2. Dilute Substrate 50 µM in Assay Buffer.

3. In a plate load 50 µL of 0.01 ng/µL rhTNAP to wells. Include a Substrate Blank of 50 µL of Assay Buffer.

4. Start the reaction by adding 50 µL of 50 µM Substrate to wells.

5. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively in kinetic mode for 5 minutes.

6. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard 4-Methumbelliferone (Sigma, Catalog # M1381).

Per Well:

• rhTNAP: 0.0005 µg

• Substrate: 25 µM

Background: Alkaline Phosphatase/ALPL

Four distinct genes encode alkaline phosphatases (APs) in humans (1). The ALPL gene encodes the liver/bone/kidney isozyme, also known as the tissue‑nonspecific AP (TNAP). In comparison, ALPI, ALPP and ALPPL2 encode intestinal, placental and placental-like or germ cell APs, respectively. The serum levels of human APs are useful tumor markers (2). There are many mutations in the ALPL gene, leading to different forms of hypophosphatasia, characterized by poorly mineralized cartilage and bones (3). The native ALPL is a glycosylated homodimer attached to the membrane through a GPI-anchor. The C-terminal pro peptide (residues 503 to 524) is not present in the mature form.

• References:

1. Le Du, M.-H. and J.L. Millan (2002) J. Biol. Chem. 277:49808.

2. Millan, J.L. and W.H. Fishman (1995) Crit. Rev. Clin. Lab. Sci. 32:1.

3. Di Mauro, S. et al. (2002) J. Bone Miner. Res. 17:1383.

• Long Name:

  Alkaline Phosphatase Liver

• Entrez Gene IDs:

  249 (Human); 11647 (Mouse)

• Alternate Names:

  Akp2; Alkaline phosphatase liver/bone/kidney isozyme; alkaline phosphatase, liver/bone/kidney; alkaline phosphatase, tissue-nonspecific isozyme; alkaline phosphomonoesterase; ALPL; APTNAP; AP-TNAP; EC 3.1.3.1; FLJ40094; FLJ93059; glycerophosphatase; HOPS; liver/bone/kidney-type alkaline phosphatase; MGC161443; tissue-nonspecific ALP; TNAP; TNSALP; TNSALPMGC167935