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Recombinant Human 6-Phosphogluconate Dehydrogenase/PGD, CF

Catalog #: 8964-DH Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
8964-DH-020
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Recombinant Human 6-Phosphogluconate Dehydrogenase/PGD, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to dehydrogenate 6-phosphogluconic acid. The specific activity is >6000 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human 6-Phosphogluconate Dehydrogenase/PGD protein
Ala2-Ala483, with a N-terminal Met and 6-His tag
Accession #
P52209
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
54 kDa
SDS-PAGE
48 kDa, reducing conditions

Product Datasheets

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8964-DH

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.

8964-DH

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, TCEP 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: 25 mM MES, 150 mM NaCl, 10 mM MgCl2, pH 7.0
  • Recombinant Human 6-Phosphogluconate Dehydrogenase/PGD (rhPGD) (Catalog # 8964-DH)
  • Donor: 6-phosphogluconic acid barium sulfate hydrate (6-PG-Ba) (Sigma, Catalog # P7627), 20 mM stock in 50 mM HCl
  • Acceptor:  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
  1. Dilute rhPGD to 1 µg/mL in Assay Buffer.
  2. Prepare reaction mixture containing 1 mM NADP+ and 0.4 mM 6-PG-Ba in Assay Buffer.
  3. Load 50 µL of 1 µg/mL rhPGD in a plate, and start the reaction by adding 50 µL of reaction mixture. Include a Control containing 50 µL of Assay Buffer and 50 µL of reaction mixture.
  4. Read at 340 nm (absorbance) in kinetic mode for 5 minutes.
  5. 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 Control
      **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:
  • rhPGD: 0.05 µg
  • NADP+: 0.5 mM
  • 6-PG-Ba: 0.2 mM
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: 6-Phosphogluconate Dehydrogenase/PGD

6-phosphogluconate dehydrogenase (PGD) is the second dehydrogenase in the pentose phosphate pathway (1). It catalyzes the oxidative decarboxylation of
6-phosphogluconate to ribulose 5-phosphate and CO2, with concomitant reduction of NADP to NADPH (2). Together with glucose-6-phosphate dehydrogenase (G6PD), these enzymes ensure the reducing environment of the cell cytoplasm (3). However, the activities of both enzymes are strongly inhibited by NADPH. It is not quite clear how the enzymes are regulated to maintain the level of reducing power inside cells (4). Deficiency of PGD is generally asymptomatic, and the inheritance of this disorder is autosomal dominant. Hemolysis results from combined deficiency of PGD and 6-phosphogluconolactonase suggest a synergism of the two enzymopathies (5).

References
  1. Barcia-Vieitez, R. and Ramos-Martinez, J.I. (2114) IUBMB Life 66:775.
  2. Wamelink, M.M. et al. (2008) J. Inherit. Metab. Dis. 31:703.
  3. Tsui, S.K.W. et al. (1996) Biochem. Genet. 34:367.
  4. Zimmer, H.G. (1996) Mol. Cell Biochem. 160-161:101.
  5. Caprari, P. et al. (2001) Ann. Hematol. 80:41.
Long Name
6-Phosphogluconate Dehydrogenase
Entrez Gene IDs
5226 (Human); 110208 (Mouse); 362660 (Rat)
Alternate Names
6PGD; EC 1.1.1.44,6-phosphogluconate dehydrogenase, decarboxylating; PGD; PGDH; phosphogluconate dehydrogenase

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