Recombinant Human Glutathione Reductase Protein, CF

Catalog #: 8866-GR Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
8866-GR-100

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Recombinant Human Glutathione Reductase Protein, CF Summary

Product Specifications

Purity

>85%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to reduce oxidized glutathione to glutathione in an NADPH-dependent manner. The specific activity is >15,000 pmol/min/μg, as measured under the described conditions.

Source

E. coli-derived human Glutathione Reductase protein
Met44-Arg522, with an N-terminal Met and 6-His tag

Accession #

P00390

N-terminal Sequence
Analysis

Met

Predicted Molecular Mass

53 kDa

SDS-PAGE

53 kDa, reducing conditions

Product Datasheets

8866-GR

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.

8866-GR

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: 0.1 M Tris, pH 7.5
Recombinant Human Glutathione Reductase (rhGSR) (Catalog # 8866-GR)
NADPH (Sigma, Catalog # N7505), 10 mM stock in deionized water
Oxidized Glutathione (Amresco, Catalog # 0524), 50 mM stock in deionized water
96-well Clear Plate (Catalog # DY990)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Create Substrate Mixture containing 0.2 mM NADPH and 1 mM Oxidized Glutathione in Assay Buffer.
Dilute rhGSR to 0.4 µg/mL in Assay Buffer.
Load 50 µL of the 0.4 µg/mL rhGSR to the plate, and start the reaction by adding 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate Mixture.
Read plate in kinetic mode for 5 minutes at an absorbance of 340 nm.
The specific activity can be calculated using the following equation:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (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 6220 M^-1cm^-1
      ***Using the path correction 0.32 cm
      Note: the output of many spectrophotometers is in mOD.

Per Well:

rhGSR: 0.02 µg
NADPH: 0.1 mM
Oxidized Glutathione: 0.5 mM

Reconstitution Calculator

Background: Glutathione Reductase

Glutathione Reductase (GR) is a homodimeric flavoprotein disulfide oxidoreductase that plays a critical role in the prevention of oxidative damage within the cell (1, 2). Glutathione Reductase helps to maintain appropriate levels of intracellular reduced glutathione (GSH) which is essential for maintaining the normal structure of red blood cells and for keeping hemoglobin in the ferrous state (3). Glutathione Reductase together with its co-factor, NADPH, catalyzes the reduction of oxidized glutathione (glutathione disulfide, GSSG) to glutathione. GSH is also a reactant for Glutathione Peroxidase, which converts hydrogen peroxide hydrogen peroxide (H2O2) into water (4). Glutathione Reductase is expressed as both mitochondrial and cytosolic isoforms, as determined by the presence or absence of an N-terminal transit peptide. Human Glutathione Reductase shares 86% and 76% amino acid sequence identity with mouse and rat Glutathione Reductase, respectively (5, 6). Alternative splicing of human Glutathione Reductase generates additional isoforms with deletions in the central pyridine nucleotide-disulfide oxidoreductase domain (7).

References