Recombinant Human Pepsinogen A5/PGA5 Protein, CF

Catalog # Availability Size / Price Qty
8457-AS-020
R&D Systems Recombinant Proteins and Enzymes
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Recombinant Human Pepsinogen A5/PGA5 Protein, CF Summary

Product Specifications

Purity
>95%, 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 cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002). The specific activity is >15,500 pmol/min/μg, as measured under the described conditions.
Source
Human embryonic kidney cell, HEK293-derived human Pepsinogen A5/PGA5 protein
Ile16-Ala388, with C-terminal 6-His tag
Accession # P0DJD9
Accession #
N-terminal Sequence
Analysis
Ile16 & Val63
Predicted Molecular Mass
41 kDa
SDS-PAGE
39-51 kDa, reducing conditions

Product Datasheets

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8457-AS

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.

8457-AS

Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
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
  • Activation Buffer: 50 mM Sodium Citrate, pH 2.5
  • Assay Buffer: 50 mM Sodium Citrate, 500 mM NaCl, 0.02% Triton® X-100, pH 4.0
  • Recombinant Human Pepsinogen A5/PGA5 (rhPGA-5) (Catalog # 8457-AS)
  • Substrate MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002), 2 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhPGA-5 to 100 µg/mL in Activation Buffer.
  2. Incubate for 15 minutes at room temperature.
  3.  Dilute activated rhPGA-5 to 0.02 µg/mL in Assay Buffer.
  4. Dilute Substrate to 20 µM in Assay Buffer.
  5. Load 50 µL of 0.02 µg/mL of rhPGA-5 into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 20 µM Substrate.
  6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  7. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)

    *Adjusted for Substrate Blank
    **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975). Per Well:

  • rhPGA-5: 0.001 µg
  • Substrate: 10 µM
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: Pepsinogen A5/PGA5

Pepsins are aspartic proteases that are synthesized in the gastric mucosa and secreted into the stomach. They are released as zymogens called pepsinogens which are then converted to active pepsins by the acidic pH of gastric juices (1-3). PGA3, PGA4, and PGA5 are human Pepsinogen A isozymogens that differ in sequence by 2­4 amino acid (aa) residues (4, 5). This recombinant human Pepsinogen A corresponds to PGA5. Human Pepsinogen A isozymogens share approximately 56% aa sequence identity with mouse and rat Pepsinogen A isozymogens. Pepsins have optimal activity under conditions of acidic pH and are inhibited by pepstatin (6, 7). Pepsin A has broad substrate specificity, but preferentially cleaves peptide bonds involving aromatic and aliphatic amino acids.

References
  1. Athauda, S.B. et al. (1989) J. Biochem. 106:920.
  2. Kageyama, T. et al. (1989) J. Biochem. 105:15.
  3. Kageyama, T. (2002) Cell. Mol. Life Sci. 59:288.
  4. Zwiers, A. et al. (1994) Clin. Nephrol. 41:153.
  5. Nakai, H. et al. (1986) Cytogenet. Cell Genet. 43:215.
  6. Marciniszyn, J., Jr. et al. (1976) J. Biol. Chem. 251:7088.
  7. Kageyama, T. and K. Takahashi (1980) J. Biochem. 88:571.
Entrez Gene IDs
5222 (Human)
Alternate Names
EC 3.4.23; EC 3.4.23.1; pepsin A; pepsinogen 5, group I (pepsinogen A); Pepsinogen A5; PGA3; PGA4; PGA5

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Fluorogenic Peptide Substrates

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