• 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 >40,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Phe18-Gly503, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Phe18

• Predicted Molecular Mass

  54 kDa

• SDS-PAGE

  77 kDa, reducing conditions

Assay Procedure

Materials

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

• Recombinant Mouse Alkaline Phosphatase/ALPL (rmALPL) (Catalog # 2910-AP)

• Substrate: 4-Methylumbelliferlyphosphate (4-MUP) (Sigma, Catalog # M8883)

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

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

1. Dilute rmALPL to 0.02 ng/µL in Assay Buffer.

2. Dilute Substrate to 50 µM in Assay Buffer.

3. Load 50 µL of 0.02 ng/µL rmALPL into a plate, and start the reaction by adding 50 µL of 50 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.

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

5. Calculate specific activity:

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

Per Well:

• rmALPL: 0.001 µg

• Substrate: 25 µM

Background: Alkaline Phosphatase/ALPL

Several distinct genes encode alkaline phosphatases (APs) in mice with different tissue-specific expression patterns. The Alpl gene, also known as Akp2, encodes the liver/bone/kidney isozyme, also known as the tissue-nonspecific AP (TNAP) (1). The Alpl gene is a key regulator of bone mineralization in mice (2). A variety of mutations in the human ALPL gene leads 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 504 to 524) is not present in the mature form.

• References:

1. Terao, M. and B. Mintz (1987) Proc. Natl. Acad. Sci. USA 84:7051.

2. Hessle, L. et al. (2002) Proc. Natl. Acad. Sci. USA 99:9445.

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