Recombinant Human PNP Protein, CF

Catalog #: 6486-NP Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
6486-NP-010

R&D Systems Recombinant Proteins and Enzymes

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All Purine Nucleoside Phosphorylase/PNP Proteins and Enzymes

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human PNP Protein, CF Summary

Product Specifications

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.

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.

Source

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

Accession #

P00491

N-terminal Sequence
Analysis

Met1

Predicted Molecular Mass

33 kDa

SDS-PAGE

30-35 kDa, reducing conditions

Product Datasheets

6486-NP

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

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.

Assay Procedure

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

Dilute rhPNP to 0.2 ng/μL in Assay Buffer.
Dilute Substrate to 800 μM in Assay Buffer.
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.
Read at an absorbance of 360 nm in kinetic mode for 5 minutes.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (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

Reconstitution Calculator

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

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

Entrez Gene IDs

4860 (Human)

Alternate Names

EC 2.4.2.1; FLJ94043; FLJ97288; Inosine phosphorylase; MGC117396; MGC125915; MGC125916; NPFLJ97312; nucleoside phosphorylase; PNP; PRO1837; PUNP; purine nucleoside phosphorylase; purine-nucleoside:orthophosphate ribosyltransferase