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 hydrolyze thymidine 5'-monophosphate p-nitrophenyl ester. The specific activity is >22,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .
Source
Human embryonic kidney cell, HEK293-derived Tyr19-Ala410, with a C-terminal 6-His tag
Accession #
N-terminal Sequence
AnalysisTyr19
Predicted Molecular Mass
45 kDa
SDS-PAGE
56-74 kDa, reducing conditions
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
Materials
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 # )
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
Background: ENPP-4
References:
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.
Long Name:
Ectonucleotide Pyrophosphatase/Phosphodiesterase 4
Entrez Gene IDs:
22875 (Human); 224794 (Mouse); 301261 (Rat)
Alternate Names:
AP3Aase; EC 3.1; ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative); E-NPP 4; ENPP4; ENPP-4; KIAA0879ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function); NPP-4; NPP4ectonucleotide pyrophosphatase/phosphodiesterase family member 4
