Recombinant Human PNP Protein, CF

R&D Systems | Catalog # 6486-NP

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human PNP Protein (6486-NP)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P00491

Applications

Enzyme Activity
View all Purine Nucleoside Phosphorylase/PNP Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human Purine Nucleoside Phosphorylase/PNP protein
Met1-Ser289, with a C-terminal 6-His tag

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met1

Predicted Molecular Mass

33 kDa

SDS-PAGE

30-35 kDa, reducing conditions

Activity

Measured by the phosphorolysis of 7-methyl-6-thioguanosine.
The specific activity is >35,000 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

6486-NP
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: Purine Nucleoside Phosphorylase/PNP

Purine Nucleoside Phosphorylase (PNP) catalyzes the phophorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. Physiological substrates of PNP include inosine, guanosine, and 2'-deoxyguanosine, but not adenosine (1). PNP is expressed in most tissues, with markedly greater expression in lymphoid tissues. Genetic deficiencies of PNP result in severely compromised T‑lymphocyte function and neurologic dysfunction (2, 3). PNP is used in assays for the measurement of inorganic phosphate (4).

References

  1. Schramm, V.L. (1998) Annu. Rev. Biochem. 67:693.
  2. Stoop, W. et al. (1977) N. Eng. J. Med. 296:651.
  3. Markert, M.L. (1991) Immunodefic. Rev. 3:45.
  4. Webb, M.R. (1992) Proc. Natl. Acad. Sci. USA. 89:4884.

Alternate Names

Inosine phosphorylase, PUNP

Entrez Gene IDs

4860 (Human)

Gene Symbol

PNP

UniProt

P00491

Additional Purine Nucleoside Phosphorylase/PNP Products

Product Documents for Recombinant Human PNP Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human PNP Protein, CF

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

For research use only

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Protocols

View specific protocols for Recombinant Human PNP Protein, CF (6486-NP):

Materials
  • Assay Buffer: 50 mM Potassium Phosphate, pH 7.4
  • Recombinant Human Purine Nucleoside Phosphorylase/PNP (rhPNP) (Catalog # 6486-NP)
  • Substrate: 7-Methyl-6-thioguanosine (MESG) (Berry & Associates, Catalog # PR 3790), 10 mM stock in DMSO
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhPNP to 0.2 ng/μL in Assay Buffer.
  2. Dilute Substrate to 800 μM in Assay Buffer.
  3. Load 50 μL of 0.2 ng/μL rhPNP into the microplate and start the reaction by adding 50 μL of Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Substrate.
  4. Read at an absorbance of 360 nm in kinetic mode for 5 minutes.
  5. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

     *Adjusted for Substrate Blank 
     **Using the extinction coefficient 6220 M-1cm-1 
     ***Using the path correction 0.320 cm
     Note: the output of many spectrophotometers is in mOD Per Well:
  • rhPNP: 0.010 μg
  • Substrate: 400 μM

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