Recombinant Human Plasma Kallikrein/KLKB1 Protein, CF Summary
Gly20-Ala638, with a C-terminal 6-His tag
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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.
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.
|Formulation||Supplied as a 0.2 μm filtered solution in Sodium Acetate 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.
- Activation Buffer: 100 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5
- Assay Buffer: 50 mM Tris, 250 mM NaCl, pH 7.5
- Recombinant Human Plasma Kallikrein/KLKB1 (rhKLKB1) (Catalog # 2497-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- EDTA (Sigma, Catalog # E-4884)
- Substrate Pro-Phe-Arg-AMC (Bachem, Catalog # I-1295)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhKLKB1 to 200 µg/mL in Activation Buffer.
- Dilute Thermolysin to 20 µg/mL in Activation Buffer.
- Combine equal volumes of 200 µg/mL rhKLKB1 and 20 µg/mL Thermolysin for final concentrations of 100 µg/mL and 10 µg/mL respectively.
- Incubate at 37 °C for 30 minutes.
- Stop the reaction with 50 µM EDTA.
- Dilute incubated rhKLKB1 to 0.5 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 μL of the 0.5 ng/µL rhKLKB1 in a plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in kinetic mode for 5 minutes.
- 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, 7-amino-4-methylcoumarin (Sigma, Catalog # A9891).
- rhKLKB1: 0.025 µg
- Substrate: 100 µM
Background: Plasma Kallikrein/KLKB1
Human 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). Human 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 human Plasma Kallikrein pro form was expressed in the NS0 cells with a foreign signal peptide. The purified enzyme is mainly the pro form. When activated by thermolysin, it displays activity against a fluorogenic peptide substrate as described in Activity Assay Protocol. This activity can be inhibited by Human Serpin G1/C1 Inhibitor (Catalog # 2488-PI).
- Coleman, R. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. (eds.) p. 1644, Academic Press, San Diego.
- Chung, N.W. et al. (1986) Biochemistry 25:2410.
Citation for Recombinant Human Plasma Kallikrein/KLKB1 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
Activation of hepatocyte growth factor activator zymogen (pro-HGFA) by human kallikrein 1-related peptidases.
Authors: Mukai S, Fukushima T, Naka D, Tanaka H, Osada Y, Kataoka H
FEBS J., 2008;275(5):1003-17.
Sample Types: Protein
Applications: Enzyme Assay
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