Recombinant Human UPP1 Protein, CF Summary
Product Specifications
Ala2-Ala310, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
7234-UP
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Glycerol. |
| 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 Procedure
- Assay Buffer: 0.1 M Potassium Phosphate, pH 7.4
- Recombinant Human Uridine Phosphorylase 1/UPP1 (rhUPP1) (Catalog # 7234-UP)
- Substrate: Uridine (Sigma, Catalog # U3003), 100 mM stock in deionized water
- UV plate (Costar, Catalog # 3635)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhUPP1 to 2 ng/μL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- Load into a plate 50 μL of 2 ng/μL rhUPP1, and start the reaction by adding 50 μL of 2 mM Substrate. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of 2 mM Substrate.
- Read plate at a wavelength of 280 nm (bottom read) in kinetic mode for 5 minutes.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Vmax* (OD/min) x -1 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 1370 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD Per Well:
- rhUPP1: 0.1 μg
- Substrate: 1 mM
Reconstitution Calculator
Background: Uridine Phosphorylase 1/UPP1
Uridine Phophorylase 1 (UPP1) belongs to the family of pentosyltransferases. UPP1 is expressed in the cytosol of most tissues and at elevated levels in many tumors (1, 2). It plays an important role in the pyrimidine salvage pathway through its catalysis of the reversible phosphorolysis of uridine to uracil (1, 2). UPP1 is important for the homeostatic regulation of intracellular and plasma uridine concentrations (3). UPP1 also plays an important role in the activation of 5-fluorouracil, an anti-tumor drug (4). The high level of UPP1 expression in some tumors makes it a potential prognostic factor for some cancers, such as oral squamous cell carcinoma (5).
- Krenitsky, T. A. et al. (1964) J. Biol. Chem. 239:805.
- Cass, C. E. and Paterson, A. R. P. (1972) J. Biol. Chem. 247:3314.
- Connolly, G.P. and Duley, J. A. (1999) Trends Pharmacol. Sci. 20:218.
- Cao, D. and Pizzorno, G. (2004) Drugs Today (Barc) 40:431.
- Miyashita, H. et al. (2002) Cancer 94:2959.
Product Specific Notices
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