Recombinant Human G6PD His-tag Protein, CF Summary
Product Specifications
Ala2-Leu515, with C-terminal 6x His tag
Product Datasheets
10096-DH
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.
10096-DH
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NADP 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: 25 mM Tris, 50 mM NaCl, 10 mM MgCl2, pH 8.0.
- Recombinant Human G6PD His-tag (rhG6PD) (Catalog # 10096-DH).
- Donor Substrate: Glucose-6-phosphate sodium salt (Sigma, Catalog # G7879), 10 mM stock in deionized water.
- Acceptor Substrate: beta -Nicotinamide adenine dinucleotide phosphate (NADP) (Sigma, Catalog # N5755), 50 mM stock in deionized water.
- 96-well Clear Plate (Catalog # DY990).
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent.
- Dilute rhG6PD to 1 ng/µL in Assay Buffer.
- Prepare substrate mixture containing 1 mM NADP and 1 mM Glucose-6-Phosphate in Assay Buffer.
- Load in a plate 50 µL of 1 ng/µL rhG6PD, 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 at 340 nm (absorbance) in kinetic mode for five minutes.
- 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) |
**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:
- rhG6PD: 0.05 µg
- NADP: 0.5 mM
- Glucose-6-phosphate: 0.5 mM
Background: G6PD
Glucose-6-phosphate dehydrogenase (G6PD) converts D-glucose 6-phosphate (G6P) into 6-phosphoglucono-δ-lactone and generate co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) (1). G6PD is the rate-limiting enzyme of the pentose phosphate pathway that supplies reducing energy to cells by maintaining the level of NADPH, which in turn maintains the level of glutathione in these cells that helps protect the red blood cells against oxidative damage from compounds like hydrogen peroxide (1, 2). More importantly, NADPH is used for biosynthesis of fatty acids or isoprenoids. G6PD is generally found as a dimer of two identical monomers (3). Depending on conditions, such as pH, these dimers can themselves dimerize to form tetramers. Each monomer in the complex has a substrate binding site that binds to G6P, and a catalytic coenzyme binding site that binds to NADP+/NADPH using the Rossman fold (4). Its activity is stimulated by the substrate G6P and NADP+. Clinically, genetic deficiency of G6PD predisposes a person to non-immune hemolytic anemia (5). G6PD is remarkable for its genetic diversity. Many variants of G6PD have been described with wide-ranging levels of enzyme activity and associated clinical symptoms. G6PD is frequently used as a coupling enzyme for measuring the enzymatic activity of glucose kinase (6).
- Au, S.W. et al. (2000). Structure 8:293.
- Thomas, D. et al. (1991). The EMBO Journal 10:547.
- Kiani, F. et al. (2007). PLOS One 2:e625.
- Kotaka, M. et al. (2005). Acta Crystallographica D 61:495.
- Cappellini, M.D. and Fiorelli, G. (2008). Lancet 371:64.
- Goward, C.R. et al. (1986) Biochemical Journal 237:415.
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