- Other Names
- ALP, Akp2, TNAP, Akp-2, APTNAP, TNSALP
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- Product Citations
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Alkaline phosphatases function physiologically as homodimers. They catalyze the hydrolysis of phosphomonoesters and release inorganic phosphate. In humans, four isoenzymes have been described: a tissue-nonspecific (TNAP) and three tissue-specific placental (PLAP), intestinal (IAP), and germ cell (GCAP). TNAP is a cell membrane-bound ecto-enzyme that releases inorganic phosphate from phosphate-enriched substrates. Also, ALPL induces the hydrolysis of extracellular inorganic pyrophosphate (PPi) which is a potent endogenous inhibitor of calcification both in vivo and in vitro. Increased activity of the enzyme alkaline phosphatase is associated with lower levels of PPi and vascular calcification present in patients with chronic kidney disease. Mutations in the TNAP gene cause the disease known as hypophosphatasia. It is characterized by poorly mineralized cartilage and bones, spontaneous fractures, and elevated concentrations of extracellular PPi.Product Details
- Mouse ALPL, amino acid Phe18 - Gly501 (Accession # BC065175) was expressed in 293E cells. The carboxy terminus contains a -6His tag.
- Molecular Mass
- The 497 amino acid recombinant protein has a predicted molecular mass of approximately 54.9 kD. The DTT-reduced and non-reduced protein migrates at approximately 75 kD by SDS-PAGE. The predicted N-terminal amino acid is Phe.
- >95%, as determined by Coomassie stained SDS-PAGE.
- 0.22 µm filtered protein solution is in 25mM HEPES pH 7.4, 150mM NaCl, 2mM MgCl2, 5µM ZnCl2.
- Endotoxin Level
- Less than 1.0 EU per µg protein as determined by the LAL method.
- 10 and 25 µg sizes are bottled at 200 µg/mL. 100 µg and larger sizes are lot-specific and bottled at the concentration indicated on the vial (please contact technical support for concentration, or use our Lookup tool if you have a lot number.)
- Storage & Handling
- Unopened vial can be stored at -20°C or -70°C for six months. For maximum results, quick spin vial prior to opening. Avoid repeated freeze/thaw cycles.
- Measured by its ability to cleave a fluorogenic substrate, 4-Methylumbelliferyl phosphate (4-MUP). The specific activity is >87,600 pmol/min/µg, as measured under the described conditions (0.001 µg/100 µL reaction).
- Application Notes
Mouse ALPL Activity Assay
- In assay buffer, prepare serial dilutions of mALPL in 1 to 2 ratios. Starting concentration is 40 ng/mL.
- In assay buffer, prepare the substrate at 50 µM.
- Add 50 µL of prepared mALPL dilutions to the assay plate.
- Begin the reaction by adding 50 µL of substrate to the plate.
- Take readings of the plate at excitation, 365nm, and emission, 445nm, wavelengths in kinetic mode for 5 minutes.
- Using the following equation, calculate the specific activity.
Specific Activity (pmol/min/µg) = [Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)]/ amount of enzyme (µg)
- mALPL amount: 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 ng
- Substrate: 25 µM
- Assay Buffer: 50 mM Tris, 1 mM MgCl2, pH 9.0
- Recombinant Mouse Alkaline Phosphatase/ALPL (mALPL)
- Substrate: 4-Methylumbelliferlyphosphate (4-MUP)
- F16 Black Maxisorp Plate
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent.
BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are validated in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at email@example.com.
Expressed in bone, liver, kidney, central nervous system, T cells, activated B cells, mesenchymal cells, fibroblasts, endothelial cells, osteoblasts, 3T3-L1 preadipocytes, murine BM preadipocytes, and embryonic stem cells.
- TNAP in bone tissue hydrolyzes inorganic pyrophosphate and promotes bone mineralization. Transcriptionally regulated by BMP2.
- Possible natural substrates: ATP, di-phosphoryl lipopolysaccharide (LPS), and phosphorylated osteopontin (p-OPN).
- Recombinant Mouse ALPL, at 0.001 µg/100 µL reaction, cleaves a fluorogenic 4-Methylumbelliferyl phosphate (4-MUP).
- Cell Type
- Fibroblasts, Leukocytes, Lymphocytes, Mesenchymal cells, Mesenchymal Stem Cells, Osteoblasts, T cells
- Biology Area
- Stem Cells
- Molecular Family
- Enzymes and Regulators
- Antigen References
- Bossi M, et al. 1993. J. Biol. Chem. 268:25409.
- Jono S, et al. 2000. J. Biol. Chem. 275:20197.
- Hessle L, et al. 2002. Proc Natl Acad Sci USA. 99:9445.
- Ciancaglini P, et al. 2010. J Bone Miner Res. 25:716.
- Kim YH, et al. 2012. Stem Cells Dev. 21:2958.
- Lei W, et al. 2013. Reproduction. 146:419.
- Narisawa S, et al. 2013. J Bone Miner Res. 28:1587.
- Millán JL and Whyte MP. 2016. Calcif Tissue Int. 98:398.
- Gene ID
- 11647 View all products for this Gene ID
- View information about ALPL on UniProt.org
- Does specific activity of a recombinant protein vary between lots?
Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.
- Have your recombinants been tested for stability?
Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.
- How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?
- Use formula Specific activity (Units/mg) = 10e6/ ED50 (ng/mL)
- How does the activity of your recombinant proteins compare to competitors?
We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!
- What is the specific activity or ED50 of my recombinant protein?
The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.