Recombinant Mouse Plasma Kallikrein/KLKB1 Protein, CF

R&D Systems | Catalog # 2498-SE

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Key Product Details

  • R&D Systems NS0-derived Recombinant Mouse Plasma Kallikrein/KLKB1 Protein (2498-SE)
  • 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

NP_032481

Structure / Form

Pro form

Applications

Enzyme Activity
View all Plasma Kallikrein/KLKB1 Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived mouse Plasma Kallikrein/KLKB1 protein
Gly20-Ala638, with a C-terminal 6-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

Gly20

Predicted Molecular Mass

70 kDa

SDS-PAGE

80 kDa, reducing conditions

Activity

Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002).
The specific activity is >300 pmol/min/µg, as measured under the described conditions.

Formulation, Preparation, and Storage

2498-SE
Formulation Supplied as a 0.2 μm filtered solution in MES and NaCl.
Shipping The product is shipped with dry ice or equivalent. 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.

Background: Plasma Kallikrein/KLKB1

Plasma kallikrein, a serine protease, is synthesized in the liver and circulates in the plasma by binding to high molecular weight (HMW) kininogen or as a free zymogen. Once activated by its physiological activator, factor XII, it displays endopeptidase activity towards peptide bonds after arginine (preferred) and lysine. It cleaves HMW kininogen, its major physiological substrate, to release the potent vasodilator peptide bradykinin. It is also able to cleave a number of inactive precursor proteins to generate active products, such as plasminogen and prourokinase. Thus, it plays an important role in blood pressure regulation, fibrinolysis, and neutrophil activation (1). Mouse plasma kallikrein precursor contains a signal peptide (residues 1 to 19) and a pro form sequence (residues 20 to 638). Upon activation, the pro form is converted to a heavy chain and a light chain, which is linked by disulfide bonds and the latter contains the catalytic domain (2). The mouse plasma kallikrein pro form was expressed in the NS0 cells with a foreign signal peptide. After being treated by thermolysin, the purified enzyme is active against a fluorogenic peptide substrate described in the Activity Assay Protocol.

References

  1. Colman, R. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. p. 1644.
  2. Seidah, N. et al. (1990) DNA and Cell Biology 9:737.

Alternate Names

KLKB1, Plasma Prekallikrein

Entrez Gene IDs

3818 (Human); 16621 (Mouse)

Gene Symbol

KLKB1

UniProt

NP_032481

Additional Plasma Kallikrein/KLKB1 Products

Product Documents for Recombinant Mouse Plasma Kallikrein/KLKB1 Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Mouse Plasma Kallikrein/KLKB1 Protein, CF

For research use only

Citations for Recombinant Mouse Plasma Kallikrein/KLKB1 Protein, CF

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Protocols

View specific protocols for Recombinant Mouse Plasma Kallikrein/KLKB1 Protein, CF (2498-SE):

Materials
  • Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5 (TCN)
  • Assay Buffer: 50 mM Tris, 250 mM NaCl, 1 mM EDTA, pH 7.5
  • Recombinant Mouse Plasma Kallikrein/KLKB1 (rmKLKB1) (Catalog # 2498-SE)
  • Activator: Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • Deactivator: EDTA  (Sigma, Catalog # E-4884)
  • Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmKLKB1 to 200 µg/mL in Activation Buffer.
  2. Dilute Thermolysin to 20 µg/mL in Activation Buffer.
  3. Activate rmKLKB1 by mixing equal volumes of diluted rmKLKB1 and Thermolysin together and incubate at 37 °C for 1 hour.
  4. After incubation, stop reaction with 50 mM EDTA (final concentration).
  5. Dilute incubated rmKLKB1 to 1 ng/µL in Assay Buffer.
  6. Dilute Substrate in Assay Buffer to 20 µM.
  7. In a plate load 50 µL of the 1 ng/µL rmKLKB1, and start the reaction by adding 50 µL 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL 20 uM Substrate without any rmKLKB1.
  8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  9. 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:

  • rmKLKB1: 0.05 µg
  • Substrate: 10 µM

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