Recombinant Human Kallikrein 1 Protein, CF Summary
Product Specifications
Ile25-Ser262, with an N-terminal human KLK-5 propeptide (Val23-Arg66) and a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
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.
2337-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and CaCl2. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5 (TCN)
- Assay Buffer: 50 mM CHES, 250 mM NaCl, pH 10.0
- 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-AMC (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
- Dilute rhKLK1 to 200 µg/mL in Activation Buffer.
- Dilute Thermolysin to 2 µg/mL in Activation Buffer.
- Combine 20 µL of diluted rhKLK1 with 20 µL of diluted Thermolysin for a final concentrations of 100 µg/mL and 1 µg/mL respectively.
- Incubate at 37 °C for 1 hour.
- Stop the reaction with 40 µL of 20 mM 1,10 Phenanthroline for a final concentration of 10 mM.
- Dilute incubated rhKLK1 to 0.4 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 µL of the 0.4 ng/µL rhKLK1 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-Methyl Coumarin (Sigma, Catalog # A-9891).
Per Well:- rhKLK1: 0.02 µg
- Substrate: 100 µM
Reconstitution Calculator
Background: Kallikrein 1
Kallikrein 1 (KLK1), also known as tissue kallikrein, is a member of human tissue kallikrein family (1). The best known physiological function of KLK1 is the cleavage of kininogen to release the vasoactive kinin peptide (bradykinin or lysyl-bradykinin), which regulates vasodilation, blood pressure reduction, smooth muscle relaxation and contraction, pain induction and inflammation (2). In addition, KLK1 may play a role in angiogensis and tumorigenesis (2). Human KLK1 precursor contains a signal peptide (residues1 to 18), a short pro peptide (residues 19 to 24) and a mature chain (residues 25 to 262). The purified rhKLK1 contains the mature chain and the propeptide from human KLK5. After being activated by thermolysin, rhKLK1 is active against a fluorogenic peptide substrate described above.
- Yousef, G.M. and E.P. Diamandis (2001) Endocrine Rev. 22:184.
- Chao, J. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. pp. 1577 - 1580.
Citations for Recombinant Human Kallikrein 1 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.
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Citations: Showing 1 - 4
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Genuine selective caspase-2 inhibition with new irreversible small peptidomimetics
Authors: E Bosc, J Anastasie, F Soualmia, P Coric, JY Kim, LQ Wang, G Lacin, K Zhao, R Patel, E Duplus, P Tixador, AA Sproul, B Brugg, M Reboud-Rav, CM Troy, ML Shelanski, S Bouaziz, M Karin, C El Amri, ED Jacotot
Cell Death & Disease, 2022-11-15;13(11):959.
Species: N/A
Sample Types: Fluorogenic Peptide Substrate
Applications: Bioassay -
Kallikrein-related peptidase 12 hydrolyzes matricellular proteins of the CCN family and modifies interactions of CCN1 and CCN5 with growth factors.
Authors: Guillon-Munos A, Oikonomopoulou K, Michel N, Smith CR, Petit-Courty A, Canepa S, Reverdiau P, Heuzé-Vourc'h N, Diamandis EP, Courty Y
J. Biol. Chem., 2011-05-31;286(29):25505-18.
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay -
Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6.
Authors: Kantyka T, Fischer J, Wu Z, Declercq W, Reiss K, Schroder J, Meyer-Hoffert U
Peptides, 2011-03-23;32(6):1187-92.
Species: Human
Sample Types: Enzyme Substrate
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Specific inhibition of tissue kallikrein 1 with a human monoclonal antibody reveals a potential role in airway diseases.
Authors: Sexton DJ, Chen T, Martik D, Kuzmic P, Kuang G, Chen J, Nixon AE, Zuraw BL, Forteza RM, Abraham WM, Wood CR
Biochem. J., 2009-08-13;422(2):383-92.
Species: Human
Sample Types: Antibody
Applications: Enzyme Assay, Surface Plasmon Resonance
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