Recombinant Human CD39L4/ENTPD5 Protein, CF Summary
Details of Functionality |
Measured by its ability to hydrolyze the 5’-phosphate groups from the substrate guanosine-5’-diphosphate (GDP). The orthophosphate product is measured by a Malachite Green Phosphate Detection Kit (Catalog # DY996). The specific activity is >7,000 pmol/min/µg, as measured under the described conditions. |
Source |
Chinese Hamster Ovary cell line, CHO-derived human CD39L4/ENTPD5 protein Val21-His428 (major) & Ser46-His428 (minor), both with a C-terminal 6-His tag Accession # O75356 |
Accession # |
|
N-terminal Sequence |
Val21 & Ser46
|
Protein/Peptide Type |
Recombinant Enzymes |
Gene |
ENTPD5 |
Purity |
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain |
Endotoxin Note |
<1.0 EU per 1 μg of the protein by the LAL method. |
Applications/Dilutions
Theoretical MW |
46 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors. |
SDS-PAGE |
50-60 kDa, reducing conditions |
Packaging, Storage & Formulations
Storage |
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.- 6 months from date of receipt, -20 to -70 °C as supplied.
- 3 months, -20 to -70 °C under sterile conditions after opening.
|
Buffer |
Supplied as a 0.2 μm filtered solution in Tris, CaCl2, NaCl and Glycerol. |
Purity |
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain |
Assay Procedure |
- Assay Buffer: 25 mM Tris, 5 mM CaCl2, pH 7.5
- Recombinant Human CD39L4/ENTPD5 (rhCD39L4) (Catalog # 5297-EN)
- Substrate: GDP (Sigma, Catalog # G-7127), 100 mM stock in deionized water
- Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhCD39L4 to 0.04 µg/mL in Assay Buffer.
- Prepare a standard curve from 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock (this is the first dilution to use as a standard).
- Perform six additional one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
- Load 25 µL of 0.04 μg/mL rhCD39L4 and the standard curve into a plate. Include a Substrate Blank containing Assay Buffer.
- Dilute the Substrate to 100 µM in Assay Buffer.
- Add 25 µL of the 100 µM Substrate to all wells and mix well.
- Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
- Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = |
Phosphate released* (nmol) x (1000 pmol/nmol) |
Incubation time (min) x amount of enzyme (µg) |
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Substrate Blank. Per Well:
- rhCD39L4: 0.001 µg
- Substrate: 35.7 µM
|
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human CD39L4/ENTPD5 Protein, CF
Background
Ectonucleoside triphosphate diphosphohydrolase-5 (NTPDase-5) is a protein that can be either secreted or reside in the lumen of the endoplasmic reticulum. The enzyme displays a specificity for nucleoside-5'-diphosphates, preferentially hydrolyzing GDP, IDP, and UDP to produce nucleoside monophosphates and orthophosphate (1). The enzyme is most highly expressed in liver, kidney, prostate, testis, and colon. NTPDase-5 (ENTPD5) and proto-oncogene CPH (PCPH) are encoded by the same gene (2). Alterations in ENTPD5/PCPH expression and mutations of ENTPD5 are associated with the progression of testicular (3), laryngeal (4), and other cancers.
- Mulero, J.J. et al. (2000) Biochemistry. 39:12924.
- Paez, J.G.. et al. (2001) Int. J. Oncol. 19:1249.
- Regadera, J. et al. (2006) Int. J. Oncol. 28:595.
- Blanquez, M.J. et al. (2002) Mol. Carcinogen. 35:186.
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