Recombinant Human POR Protein, CF Summary
Product Specifications
Arg45-Ser677, with N-terminal amino acids WAGALA 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.
6340-PR
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and DTT. |
| 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: 0.3 M KH2PO4, pH 8.0
- Recombinant Human POR/Cytochrome P450 Reductase (rhPOR) (Catalog # 6340-PR)
- Substrate: beta -NADPH (Sigma, Catalog # N7505), 10 mM stock in deionized water (8.33 mg/mL)
- Cytochrome C, Bovine heart (Sigma, Catalog # C3131), 2 mg/mL stock in deionized water
- 96 well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhPOR to 0.2 ng/μL in Assay Buffer.
- Combine 480 μL of Assay Buffer, 500 μL of 2 mg/mL Cytochrome C, and 20 μL of 10 mM beta -NADPH.
- Load 50 μL of 0.2 ng/μL rhPOR and start the reaction by adding 50 μL of Substrate Mixture. Include a Substrate Blank containing 50 μL Assay Buffer and 50 μL Substrate Mixture.
- Read in kinetic mode for 6 minutes with 1 minute lag time at an absorbance of 550 nm.
- 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 21100 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD Per Well:
- rhPOR: 0.010 μg
- Cytochrome C: 0.5 mg/ml
- beta -NADPH: 0.1 mM
Reconstitution Calculator
Background: POR/Cytochrome P450 Reductase
NADPH-Cytochrome P450 Reductase (P450R) is an essential component of the cytochrome P450 monooxygenase system of eukaryotic cells (1). P450R is anchored in the endoplasmic reticulum membrane with its catalytic domain residing in the cytosol. P450R is a flavoprotein, containing one molecule each of FMN and FAD, which are essential for the transfer of electrons from NADPH to the cytochromes P450 (2). This reduction is necessary for cytochromes P450 to perform each cycle of oxidation. P450R is also capable of transferring electrons to cytochrome b5, heme oxygenase, the fatty acid elongation system, and other proteins. Mutations of P450R can result in disordered steroidogenesis and Antley-Bixler syndrome.
- Philips, A.H. and R.G. Langdon (1962) J. Biol. Chem. 237:2652.
- Iyanagi, T. and H.S. Mason (1973) Biochemistry 12:2291.
- Flueck C.E. et al. (2004) Nat. Genet. 36:228.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.FAQs
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Reason for Rating: I have compared R&D's POR with POR from other resources for my enzymatic activity assay, this is the only one that works, thank you very much.
