Recombinant Mouse ENPP-4 Protein, CF Summary
Product Specifications
Tyr19-Ala410, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
8996-EN
| 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 Procedure
- Assay Buffer: 50 mM Tris, pH 7.5
- Recombinant Mouse ENPP-4 (rmENPP-4) (Catalog # 8996-EN)
- Substrate: Thymidine 5’-monophosphate p-nitrophenyl ester (Sigma, Catalog # T4510), 100 mM stock in deionized water
- NaOH, 0.2 M in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rmENPP-4 to 0.1 ng/µL in Assay Buffer.
- Dilute Substrate to 10 mM in Assay Buffer.
- Load 50 µL of 0.1 ng/µL rmENPP-4 in a clear strip well plate, and start the reaction by adding 50 µL of 10 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL 10 mM Substrate.
- Incubate sealed plate at room temperature for 30 minutes.
- Stop reactions by adding 100 µL of 0.2 M NaOH to all wells, including Substrate Blank wells.
- Read at 410 nm (absorbance) in endpoint mode.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Abs* (OD) x Conversion Factor** (pmol/OD) |
| Incubation time (min) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard p-Nitrophenol (Sigma, Catalog # 241326). Per Reaction:- rmENPP-4: 0.005 µg
- Substrate: 5 mM
Reconstitution Calculator
Background: ENPP-4
Ectonucleotide pyrophosphatase/phosphodiesterase 4 (ENPP-4 or NPP4) belongs to a group of ecto-enzymes which regulate the availability of extracellular nucleotides (1). This enzyme family forms a subgroup of a larger family that also includes arylsulfatases, phosphopentomutases, 2,3-bisphosphoglycerate-independent phosphoglycerate mutases (iPGM), and alkaline phosphatases (2). Mature mouse ENPP-4 consists of a 392 amino acid (aa) ectodomain that contains the catalytic domain with a zinc-coordinated substrate binding pocket, a 21 aa transmembrane segment, and a 25 aa cytoplasmic tail (3). It shares 86% and 90% aa sequence identity with human and rat ENPP-4, respectively. Alternative splicing generates a short isoform with a 32 aa deletion in the phosphodiesterase domain. ENPP-4 hydrolyzes phosphodiester bonds in nucleotides with a preference for adenine nucleotides (3). It cleaves the diadenosine compounds Ap3A and Ap4a which are released from the dense granules of thrombin-activated platelets (3, 4). These reactions generate AMP and ADP from Ap3A cleavage, and AMP and ATP from Ap4A cleavage (4). ENPP-4 is expressed on the surface of vascular endothelial cells where its activity prolongs platelet aggretation and contributes to thrombus formation (4).
- Zimmermann, H. et al. (2012) Purinergic Signal. 8:437.
- Gijsbers, R. et al. (2001) J. Biol. Chem. 276:1361.
- Albright, R.A. et al. (2014) J. Biol. Chem. 289:3294.
- Albright, R.A. et al. (2012) Blood 120:4432.
Citation for Recombinant Mouse ENPP-4 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Identification of the extracellular membrane protein ENPP3 as a major cGAMP hydrolase and innate immune checkpoint
Authors: Mardjuki, R;Wang, S;Carozza, J;Zirak, B;Subramanyam, V;Abhiraman, G;Lyu, X;Goodarzi, H;Li, L;
Cell reports
Species: N/A
Sample Types: Small Molecule
Applications: Bioassay
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