Recombinant Mouse Alkaline Phosphatase/ALPL Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Alkaline Phosphatase/ALPL Protein, CF Summary

• Details of Functionality: 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.
• Source: Mouse myeloma cell line, NS0-derived mouse Alkaline Phosphatase/ALPL protein
  Phe18-Gly503, with a C-terminal 6-His tag
• Accession #: P09242
• N-terminal Sequence: Phe18
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Alpl
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 54 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 77 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • 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:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

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

  Per Well:
  • rmALPL: 0.001 µg
  • Substrate: 25 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Alkaline Phosphatase/ALPL Protein, CF

• 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

Background

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.

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.

Bioinformatics

• Gene Symbol: Alpl
• Uniprot:
  • P09242()