Recombinant Human Kallikrein 4/Prostase Protein, CF Summary
Ser27-Ser254, with a C-terminal 10-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||Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.|
|Reconstitution||Reconstitute at 200 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.
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5 (TCN)
- Assay Buffer: 50 mM Tris, pH 9.0
- Recombinant Human Kallikrein 4/Prostase/EMSP1 (rhKLK4) (Catalog # 1719-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
- Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011), 90 mM Stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhKLK4 to 200 µg/mL in Activation Buffer.
- Dilute Thermolysin to 2 µg/mL in Activation Buffer.
- Combine equal volumes diluted rhKLK4 and Thermolysin and incubate at 37 °C for 2 hours to activate.
- Stop the reaction with 1,10 Phenanthroline at a final concentration of 10 mM.
- Dilute activated rhKLK4 to 2 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 µL of the 2 ng/μL rhKLK4 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 without any rhKLK4.
- 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).
- rhKLK4: 0.100 µg
- Substrate: 100 µM
Background: Kallikrein 4/Prostase/EMSP1
Kallikrein 4 (KLK4), also known as prostase or enamel matrix serine protease 1 (EMSP1), is a serine protease of the human tissue kallikrein gene family (1). Among normal tissues, human KLK4 is specifically expressed in the prostate (2). It is over-expressed in prostate cancer and this expression is regulated by hormones including androgens, esterogen and progesterone (3). rhKLK4 readily activates pro-KLK3/PSA and pro-urokinase type plasminogen activator (uPA), indicating it may initiate events involving PSA and uPA in either normal or abnormal processes (4). KLK4 may have additional roles such as functioning as one of the two major enamel proteases identified that process enamel matrix proteins (5). In addition to being a secreted enzyme, it is also a nuclear protein (3, 6). The deduced amino acid sequence of human KLK4 consists of a signal peptide, a short pro region and a mature/active enzyme. rhKLK4 can be activated in vitro by thermolysin, a zinc protease. The peptidase activity can be inhibited by AEBSF (R&D Systems, Catalog # EI001), a general serine protease inhibitor.
- Yousef, G.M. and E.P. Diamandis (2001) Endocrine Rev. 22:184.
- Nelson, P.S. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3114.
- Xi, Z. et al. (2004) Cancer Res. 64:2365.
- Takayama, T.K. et al. (2001) Biochemistry 40:15341.
- Simmer, J.P. and J.C. Hu (2002) Connect Tissue Res. 43:441.
- Ryu, O.H. et al. (2002) Eur. J. Oral. Sci. 110:358.
Citations for Recombinant Human Kallikrein 4/Prostase 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.
Citations: Showing 1 - 5
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Pathogenesis of Molar Hypomineralisation: Hypomineralised 6-Year Molars Contain Traces of Fetal Serum Albumin
Authors: R Williams, VA Perez, JE Mangum, MJ Hubbard
Front Physiol, 2020;11(0):619.
Sample Types: Protein
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;422(2):383-92.
Sample Types: Antibody
Applications: Surface Plasmon Resonance
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
Activation profiles and regulatory cascades of the human kallikrein-related peptidases.
Authors: Yoon H, Laxmikanthan G, Lee J, Blaber SI, Rodriguez A, Kogot JM, Scarisbrick IA, Blaber M
J. Biol. Chem., 2007;282(44):31852-64.
Sample Types: Peptide
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
Sample Types: Enzyme Substrate
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Fluorogenic Peptide Substrates
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