Recombinant Human Kininogen HMW (HKa) Protein, CF

R&D Systems | Catalog # 1569-PI

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human Kininogen HMW (HKa) Protein (1569-PI)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

NS0

Accession Number

P01042

Structure / Form

High molecular weight form consisting of the mature chain, the heavy and light chains, and a minor species

Applications

Inhibition Activity
View all Kininogen Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human Kininogen protein
Gln19-Ser644, with an N-terminal signal sequence and a C-terminal 10-His tag

Purity

>95%, 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.

N-terminal Sequence Analysis

Ser390 (light chain) & Lys438 (minor species) with Gln19 predicted to be present and blocked

Predicted Molecular Mass

71 kDa (mature), 41 kDa (heavy) & 30 kDa (light)

SDS-PAGE

120 kDa, 60 kDa and 48 kDa, reducing conditions

Activity

Measured by its ability to inhibit papain cleavage of a fluorogenic peptide substrate Z-FR-AMC (Catalog # ES009).
The IC50 value is <3 nM, as measured under the described conditions.

Formulation, Preparation, and Storage

1569-PI
Formulation Lyophilized from a 0.2 μm filtered solution in Sodium Acetate and NaCl.
Reconstitution Reconstitute at 100 μg/mL in sterile 25 mM Tris and 100 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.

Calculators

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: Kininogen

Kininogen, also known as alpha 2-thiol proteinase inhibitor, is a multi-function protein. There are two alternatively spliced forms, designated as the high molecular weight (HMW) and low MW (LMW) forms (1). The HMW form is synthesized as a 644 amino acid residue precursor with a signal peptide (residues 1‑18). The mature chain (residues 19‑644) is further processed into the heavy (residues 19‑380) and the light (residues 390‑644) chains. The active peptide bradykinin (residues 381‑389) is released, which has a variety of functions including muscle contraction, hypotension and inflammation. The heavy chain consists of three cystatin‑like domains, which are responsible for inhibiting cysteine proteases. The light chain consists of a His-rich domain, which is associated with the clotting activity. In comparison to the HMW form, the LMW Kininogen (427 residues) has the same sequence in its heavy chain and bradykinin, but a different sequence in its light chain (residues 402‑427). The LMW form is not involved in blood clotting.

References

  1. Takagaki, Y. et al. (1985) J. Biol. Chem. 260:8601.

Alternate Names

alpha 2-thiol Proteinase Inhibitor, BDK, KNG1

Entrez Gene IDs

3827 (Human); 16644 (Mouse); 24903 (Rat)

Gene Symbol

KNG1

UniProt

P01042

Additional Kininogen Products

Product Documents for Recombinant Human Kininogen HMW (HKa) Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human Kininogen HMW (HKa) Protein, CF

For research use only

Citations for Recombinant Human Kininogen HMW (HKa) Protein, CF

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Protocols

View specific protocols for Recombinant Human Kininogen HMW (HKa) Protein, CF (1569-PI):

Materials
  • Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
  • Assay Buffer: 50 mM Tris, pH 7.0
  • Recombinant Human Kininogen High Molecular Weight (HKa) (rhKininogen) (Catalog # 1569-PI)
  • Papain (Sigma, Catalog # P4762)
  • Substrate: Z-Phe-Arg-AMC (R&D Systems, Catalog  # ES009), 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. Chill Activation Buffer on ice.
  2. Dilute Papain to 100 µg/mL in Activation Buffer.
  3. Incubate at room temperature for 10 minutes.
  4. Prepare a dilution curve of rhKininogen (MW: 71,211Da) in Assay Buffer. Make the following serial dilutions: 400, 200, 100, 50, 25, 12.5, 6.25, and 3.125 nM.
  5. Dilute activated Papain to 2 µg/mL in Assay Buffer.
  6. Mix equal volumes of the rhKininogen curve dilutions and the diluted active Papain. Include a control (in duplicate) containing Assay Buffer and the diluted active Papain.
  7. Incubate mixtures at room temperature for 10 minutes.
  8. Dilute Substrate to 200 µM in Assay Buffer.
  9. Perform a 1/5 dilution with Assay Buffer to the incubated mixture of rhKininogen and Papain.
  10. Load 50 µL of diluted incubated mixture into the plate, and start the reaction by adding 50 µL of 200 µM Substrate.
  11. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
  12. Derive the 50% inhibition concentration (IC50) for rhKininogen by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  13. The specific activity for Papain 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 (AMC) (Sigma, Catalog # A-9891).

Per Well:
  • Papain: 0.010 µg
  • Substrate: 100 µM
  • rhKininogen curve: 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 nM

FAQs

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Associated Pathways

Blood Coagulation Signaling Pathways Blood Coagulation Signaling Pathway Thumbnail