Recombinant Mouse Alkaline Phosphatase/ALPL Protein, CF
Recombinant Mouse Alkaline Phosphatase/ALPL Protein, CF Summary
Phe18-Gly503, with a C-terminal 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- 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
- Dilute rmALPL to 0.02 ng/µL in Assay Buffer.
- Dilute Substrate to 50 µM in Assay Buffer.
- 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.
- Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in kinetic mode for 5 minutes.
- 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).
- 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.
- Terao, M. and B. Mintz (1987) Proc. Natl. Acad. Sci. USA 84:7051.
- Hessle, L. et al. (2002) Proc. Natl. Acad. Sci. USA 99:9445.
- Di Mauro, S. et al. (2002) J. Bone Miner. Res. 17:1383.
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