Recombinant Human POR/Cytochrome P450 Reductase Protein, CF

Catalog #: 6340-PRB Datasheet / COA / SDS

His-tag

Catalog #	Availability	Size / Price	Qty
6340-PRB-020

2 Images

All POR/Cytochrome P450 Reductase Proteins and Enzymes

Product Details

Reviews

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

Recombinant Human POR/Cytochrome P450 Reductase Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

Activity

Measured by the reduction of cytochrome c using NADPH as the cofactor. The specific activity is >1800 pmol/min/μg, as measured under the described conditions.

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived human POR/Cytochrome P450 Reductase protein
Ser62-Ser677 with an N-terminal Met and 6-His tag

Accession #

P16435.2

N-terminal Sequence
Analysis

Met

Predicted Molecular Mass

71 kDa

SDS-PAGE

63-69 kDa, under reducing conditions.

Product Datasheets

6340-PRB

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.

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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

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 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 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: 100 μM

Scientific Data

Enzyme Activity

View Larger

Recombinant Human POR His-tag (Catalog # 6340-PRB) is measured by the reduction of cytochrome c using NADPH as the cofactor.

SDS-PAGE

View Larger

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).

References

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.

Long Name

NADPH Cytochrome P450 Reductase

Entrez Gene IDs

5447 (Human)

Alternate Names

CPR; CYPOR; CYPORFLJ26468; Cytochrome P450 Reductase; DKFZp686G04235; EC 1.6.2.4; NADPH--cytochrome P450 reductase; NADPH-dependent cytochrome P450 reductase; P450 (cytochrome) oxidoreductase; P450R; POR