Recombinant Human PNP Protein, CF Summary
Met1-Ser289, with a C-terminal 6-His tag
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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.
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.
|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.
- 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 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
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).
- 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.
Product Specific NoticesCoomassie is a registered trademark of Imperial Chemical Industries Ltd.
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