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Recombinant Human Serpin A5 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Inhibition Activity
Format
Carrier-Free

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Recombinant Human Serpin A5 Protein, CF Summary

Details of Functionality
Measured by its ability to inhibit Recombinant Human Coagulation Factor II/Thrombin (Catalog # 1473-SE) cleavage of a fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011). The IC50 value is <2 nM, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Serpin A5/Protein C Inhibitor protein
His20-Pro406, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
His20
Protein/Peptide Type
Recombinant Enzymes
Gene
SERPINA5
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Dilutions
  • Inhibition Activity
Theoretical MW
45 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
50-55 kDa, reducing conditions
Publications
Read Publications using
1266-PI in the following applications:

Packaging, Storage & Formulations

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 reconstitution.
Buffer
Lyophilized from a 0.2 μm filtered solution in MES and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Reconstitution Instructions
Reconstitute at 100 μg/mL in sterile 25 mM MES, 150 mM NaCl, pH 6.5.
Assay Procedure
  • Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Human Serpin A5/Protein C Inhibitor (rhSerpin A5) (Catalog # 1266-PI)
  • Recombinant Human Coagulation Factor II/Thrombin (Catalog # 1473-SE)
  • Heparin (Sigma, Catalog # H3393), 20 mg/mL stock in deionized water
  • Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute Thrombin to 0.4 µg/mL with 48.6 µg/mL Heparin in Assay Buffer.
  2. Prepare a curve of rhSerpin A5 (MW: 45,003 Da) in Assay Buffer. Make the following serial dilutions:  200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, and 0.391 nM.
  3. Mix equal volumes of rhSerpin A5 curve dilutions and Thrombin/Heparin mixture. Include a control (in duplicate) containing equal volumes of Assay Buffer and diluted Thrombin/Heparin mixture.
  4. Incubate reaction mixtures at room temperature for 30 minutes.
  5. After incubation, dilute the reaction mixtures by 1/5 in Assay Buffer.
  6. Dilute Substrate to 200 µM in Assay Buffer.
  7. In a plate load 50 µL of the diluted reaction mixtures to wells, and start the reaction by adding 50 µL of 200 µM Substrate.
  8. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  9. Derive the 50% inhibiting concentration (IC50) value by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  10. The specific activity for Thrombin at each point may be determined using the following formula (if needed):

     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 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

Per Well:
  • Thrombin: 0.002 µg
  • rhSerpin A5: 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 nM
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Serpin A5 Protein, CF

  • Acrosomal serine protease inhibitor
  • antitrypsin), member 5
  • member 5
  • PAI-3
  • PAI3PLANH3
  • PCI
  • PCIplasminogen activator inhibitor III
  • Plasminogen activator inhibitor 3
  • plasminogen activator inhibitor-3
  • PROCIplasma serine protease inhibitor
  • Protein C inhibitor
  • serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase
  • Serpin A5
  • serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin)
  • SERPNA5

Background

Serpin A5 is the a member of the Serpin superfamily and inhibits a variety of serine proteases such as protein C, plasminogen activators, thrombin, factor Xa, several kallikreins and acrosin (1). Serpin A5 is synthesized in the liver and secreted in plasma. It is found in numerous steroid-responsive organs and has been detected in saliva, cerebral spinal fluid, amniotic fluid, tears and semen. Because of its protease targets and regulated expression patterns, Serpin A5 has been proposed to play a role in processes such as blood coagulation, fertilization and carcinogenesis (2, 3). Similar to Serpins C1 and D1, its thrombin inhibitory activity is enhanced by heparin.

  1. Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
  2. Palmier, D. et al. (2002) J. Biol. Chem. 277:40950.
  3. Geiger, M. et al. (1996) Immunopharmacology 32:53.

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Publications for Serpin A5/Protein C Inhibitor (1266-PI)(2)

We have publications tested in 1 confirmed species: Human.

We have publications tested in 1 application: Enzyme Assay.


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Bioinformatics

Gene Symbol SERPINA5
Uniprot