• 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    
  Analysis

  Tyr19

• Predicted Molecular Mass

  45 kDa

• SDS-PAGE

  56-74 kDa, reducing conditions

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

1. Dilute rmENPP-4 to 0.1 ng/μL in Assay Buffer.

2. Dilute Substrate to 10 mM in Assay Buffer.

3. 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.

4. Incubate sealed plate at room temperature for 30 minutes.

5. Stop reactions by adding 100 μL of 0.2 M NaOH to all wells, including Substrate Blank wells.

6. Read at 410 nm (absorbance) in endpoint mode.

7. Calculate specific activity:

     *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:

1.  Zimmermann, H. et al. (2012) Purinergic Signal. 8:437.

2. Gijsbers, R. et al. (2001) J. Biol. Chem. 276:1361.

3. Albright, R.A. et al. (2014) J. Biol. Chem. 289:3294.

4. 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