Recombinant Human Serpin A4/Kallistatin Protein, CF

Catalog # Availability Size / Price Qty
1669-PI-020
Product Details
Citations (1)
FAQs
Supplemental Products
Reviews (3)

Recombinant Human Serpin A4/Kallistatin Protein, CF Summary

Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
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
Analysis
No results obtained: Gln21 predicted
Predicted Molecular Mass
48 kDa
SDS-PAGE
57 kDa and 61 kDa, reducing conditions

Product Datasheets

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.

1669-PI

Formulation Lyophilized from a 0.2 μm filtered solution in MES and NaCl.
Reconstitution Reconstitute at 100 μg/mL in sterile 50 mM Tris, 10 mM CaCl2 and 150 mM NaCl, pH 7.5.
Shipping The product is shipped at ambient temperature. 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 reconstitution.

Assay Procedure

Materials
  • 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
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: Serpin A4/Kallistatin

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).

References
  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.
Entrez Gene IDs
5267 (Human)
Alternate Names
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)

Citation for Recombinant Human Serpin A4/Kallistatin Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

1 Citation: Showing 1 - 1

  1. Enzymatic properties of human kallikrein-related peptidase 12 (KLK12).
    Authors: Memari&lt;/LastName&gt;&lt;ForeNam N&lt;/Initial, Memari N, Jiang W, Diamandis EP, Luo LY
    Biol. Chem., 2007;388(4):427-35.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Enzyme Assay

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Reviews for Recombinant Human Serpin A4/Kallistatin Protein, CF

Average Rating: 4.7 (Based on 3 Reviews)

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Recombinant Human Serpin A4/Kallistatin Protein, CF
By Anonymous on 07/02/2018
Application: Enzymatic activity in vitro

Recombinant Human Serpin A4/Kallistatin Protein, CF
By Anonymous on 06/21/2018
Application: Specificity, inhibition kinetic assay

Recombinant Human Serpin A4/Kallistatin Protein, CF
By Anonymous on 03/23/2018
Application: Other