Recombinant Human Serpin A4/Kallistatin 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 A4/Kallistatin Protein, CF Summary

Details of Functionality
Measured by its ability to inhibit KLK1 cleavage of a fluorogenic peptide substrate, Pro-Phe-Arg-7-amido-4-methylcoumarin (PFR-AMC). The IC50 value is <40 nM, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Serpin A4/Kallistatin protein
Gln21-Pro427, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
No results obtained: Gln21 predicted
Protein/Peptide Type
Recombinant Enzymes
Gene
SERPINA4
Purity
>90%, 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
48 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
57 kDa and 61 kDa, reducing conditions
Publications
Read Publication using
1669-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
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Reconstitution Instructions
Reconstitute at 100 μg/mL in sterile 50 mM Tris, 10 mM CaCl2 and 150 mM NaCl, pH 7.5.
Assay Procedure
  • Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5 (TCN)
  • Inhibition Buffer: 25 mM Tris, 150 mM NaCl, pH 7.5
  • Assay Buffer: 50 mM CHES, 250 mM NaCl, pH 10.0
  • Recombinant Human Serpin A4/Kallistatin (rhSerpin A4) (Catalog # 1669-PI)
  • Recombinant Human Kallikrein 1 (rhKLK1) (Catalog # 2337-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • 1,10-Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
  • Substrate:  Pro-Phe-Arg-7-amido-4-methylcoumarin (Bachem, Catalog # I-1295), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Activate rhKLK1 at 100 µg/mL with 1 µg/mL Thermolysin in Activation Buffer.
  2. Incubate at 37 °C for 1 hour.
  3. Add 1,10-Phenathroline at a final concentration of 10 mM to stop activation reaction.
  4. Prepare a curve of rhSerpin A4 (47710 Da) in Inhibition Buffer. Make the following serial dilutions: 8000, 4000, 2000, 1000, 625, 415, 200, and 75 nM.
  5. Dilute activated rhKLK1 to 5 µg/mL in Inhibition Buffer.
  6. Combine 20 µL of 5 µg/mL rhKLK1 with 20 µL of rhSerpin A4 curve dilutions. Include two controls of 20 µL Inhibition Buffer with 20 µL of 5 µg/mL rhKLK1.
  7. Incubate curve at 37 °C for 1 hour.
  8. Dilute curve 12.5 fold by adding 460 µL of Assay Buffer to each dilution.
  9. Dilute Substrate to 200 µM in Assay Buffer.
  10. In a plate load 50 µL of the rhSerpin A4 curve, and start the reaction by adding 50 µL of 200 µM Substrate to wells.
  11. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  12. Derive the 50% inhibiting concentration (IC50) of rhSerpin A4 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  13. The specific activity for rhKLK1 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:
  • rhKLK1: 0.010 µg
  • rhSerpin A4 curve: 160, 80, 40, 20, 12.5, 8.3, 4 and 1.5 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 A4/Kallistatin Protein, CF

  • antitrypsin), member 4
  • KAL
  • Kallistatin
  • KLST
  • KSTKallikrein inhibitor
  • member 4
  • PI-4
  • PI4Peptidase inhibitor 4
  • protease inhibitor 4 (kallistatin)
  • serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase
  • Serpin A4
  • serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin)

Background

Serpin A4 is a member of the Serpin superfamily of the serine protease inhibitors (1). Known also as kallistatin or proteinase inhibitor 4, Serpin A4 has been shown to function as a specific inhibitor of tissue kallikrein and as an inhibitor of angiogenesis and tumor growth (2, 3).

  1. Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
  2. Chen, V.C. et al. (2000) J. Biol. Chem. 275:40371.
  3. Miao, R.Q. et al. (2002) Blood 100:3245.

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Publications for Serpin A4/Kallistatin (1669-PI)(1)

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 SERPINA4
Uniprot