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Recombinant Human 15-PGDH/HPGD Protein, CF

Catalog #: 5660-DH Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
5660-DH-010
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Recombinant Human 15-PGDH/HPGD Protein, 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
Measure by the production of NADH during the oxidation of PGF2 alpha. The specific activity is >1,500 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived human 15-PGDH/HPGD protein
Met1-Gln266, with a C-terminal 6-His tag
Accession #
P15428
N-terminal Sequence
Analysis
Met1
Structure / Form
Noncovalently-linked homodimer
Predicted Molecular Mass
30 kDa
SDS-PAGE
28 kDa, reducing conditions

Product Datasheets

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5660-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.

5660-DH

Formulation Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT and Glycerol.
Shipping The product is shipped with dry ice or equivalent. 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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Assay Buffer: 50 mM Tris, 100 mM NaCl, 2 mM Dithiothreitol (DTT), pH 9.0
  • Recombinant Human 15‑PGDH/HPGD (rhHPGD) (Catalog # 5660-DH)
  • beta -Nicotinamide adenine dinucleotide ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
  • Prostaglandin F2 alpha (PGF2 alpha ) (Sigma, Catalog # P0424), 10 mM stock in absolute ethanol
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare the Substrate Mixture.
    1. Dilute beta -NAD to 1 mM in Assay Buffer.
    2. Dilute PGF2 alpha to 0.4 mM in Assay Buffer.
    3. Mix equal volumes of each for a final concentration of 0.5 mM beta -NAD and 0.2 mM PGF2 alpha.
  2. Dilute rhHPGD to 1.0 ng/μL in Assay Buffer.
  3. Load in a plate 50 μL of 1.0 ng/μL rhHPGD, and start the reaction by adding 50 μL of Substrate Mixture.
  4. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
  5. Read at 339 nm in kinetic mode for 5 minutes.

     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 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:
  • rhHPGD: 0.050 µg
  • beta -NAD: 0.25 mM
  • PGF2 alpha : 0.1 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: 15-PGDH/HPGD

HPGD, or 15-hydroxyprostaglandin dehydrogenase, is a NAD+-linked dehydrogenase that oxidizes the hydroxyl group at position 15 of prostaglandins to a ketone, resulting in a loss of biological activity (1). HPGD is a major enzyme for the catabolism of prostaglandins. The enzyme is a member of the short-chain alcohol dehydrogenase family of enzymes (2). HPGD is a cytosolic enzyme expressed in most tissues, with highest expression levels in placenta, lung, and kidney (3). It is inhibited by aspirin and nonsteroidal anti-inflammatory drugs (4). Defects in HPGD are a cause of hypertrophic osteoarthropathy (5).

References
  1. Anggard, E. and B. Samuelsson (1964) J. Biol. Chem. 239:4097.
  2. Krook, M. et al. (1990) Biochemistry 29:738.
  3. Tai, H.H. (1976) Biochemistry 15:4586.
  4. Mak, O.T. et al. (1982) Biosci. Rep. 2:503.
  5. Uppal, S. et al. (2008) Nat. Genet. 40:789.
Long Name
15-Hydroxyprostaglandin Dehydrogenase
Entrez Gene IDs
3248 (Human); 15446 (Mouse); 79242 (Rat)
Alternate Names
15-hydroxyprostaglandin dehydrogenase [NAD+]; 15PGDH; 15-PGDH; EC 1.1.1; EC 1.1.1.141; HPGD; hydroxyprostaglandin dehydrogenase 15-(NAD); NAD+-dependent 15-hydroxyprostaglandin dehydrogenase; PGDH1; PGDH1PGDH; Prostaglandin dehydrogenase 1; SDR36C1; short chain dehydrogenase/reductase family 36C, member 1

FAQs

  1. Why am I observing an additional higher molecular weight band?

    • We expect this higher molecular weight band to be the homodimer. In this homodimer, the two monomers are noncovalently associated with each other. Since they are not joined by disulfide bonds, reducing conditions are not expected to separate the two monomers.

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