Recombinant Human POR/Cytochrome P450 Reductase Protein, CF
Recombinant Human POR/Cytochrome P450 Reductase Protein, CF Summary
Product Specifications
Ser62-Ser677 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.
6340-PRB
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Glycerol. |
| Shipping | The product is shipped with polar packs. 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 Potassium Phosphate, pH 8.0
- Recombinant Human POR/Cytochrome P450 Reductase His-tag (rhPOR) (Catalog # 6340-PRB)
- Substrate: beta -NADPH, 10 mM stock in deionized water
- Cytochrome C, Bovine heart, 2 mg/mL stock in deionized water
- Clear 96 well Plate (Catalog # DY990)
- Plate Reader with Absorbance Read Capability
- Dilute rhPOR to 0.2 μg/mL in Assay Buffer.
- Prepare a Substrate Mixture containing 200 µM beta -NADPH and 1 mg/mL Cytochrome C in Assay Buffer.
- Load 50 μL of 0.2 μg/mL 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) |
- rhPOR: 0.010 μg
- Cytochrome C: 0.5 mg/ml
- beta -NADPH: 100 μM
Scientific Data
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Recombinant Human POR His-tag (Catalog # 6340-PRB) is measured by the reduction of cytochrome c using NADPH as the cofactor.
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2 μg/lane of Recombinant Human POR/Cytochrome P450 Reductase His-tag Protein (Catalog # 6340-PRB) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 63-69 kDa, under reducing conditions.
Reconstitution Calculator
Background: POR/Cytochrome P450 Reductase
NADPH-Cytochrome P450 Reductase (P450R), or POR, 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. The protein is composed of four domains: an N-terminal FMN‑binding domain, a connecting domain, and C-terminal FAD- and NADPH-binding domains (2). The flavoprotein P450R contains one molecule each of FMN and FAD, which are essential for the transfer of electrons from NADPH to the P450 cytochromes (3). This reduction is necessary for cytochromes P450 to perform each cycle of oxidation. P450R plays a vital role in metabolism and detoxification of xenobiotic and endobiotic compounds (4). P450R is also capable of transferring electrons to cytochrome b5, heme oxygenase, the fatty acid elongation system, and other proteins (4). Mutations of P450R can result in disordered steroidogenesis and bone defects in Antley-Bixler syndrome (5, 6) and expression of P450R has been correlated with patient outcome in breast cancer (7).
- Philips, A.H. and R.G. Langdon (1962) J. Biol. Chem. 237:2652.
- Wang, M. et al. (1997) Proc. Natl. Acad. Sci. 94:8411.
- Iyanagi, T. and H.S. Mason (1973) Biochemistry 12:2291.
- Iyanagi, T. (2007) Int. Rev. Cytol. 260:35.
- Flueck C.E. et al. (2004) Nat. Genet. 36:228.
- Polusani, S.R. et al. (2011) Biochem. Biophys. Res. Commun. 411:490.
- Pederson, M.H. et al. (2019) Int. J. Cancer 144:631.
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