>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
<1.0 EU per 1 μg of the protein by the LAL method.
26 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011), 100 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate reader (Model: SpectramMax Gemini EM by Molecular Devices) or equivalent
Dilute rhKLK14 to 200 µg/mL in Activation Buffer.
Dilute Thermolysin to 20 µg/mL in Activation Buffer.
Combine 25 µL of diluted rhKLK14 and 25 µL of diluted Thermolysin.
Incubate at 37 °C for 1 hour.
Stop reaction with 50 µL of 100 mM EDTA. Dilute incubated rhKLK14 mixture to 0.2 ng/µL in Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer.
Load 50 µL of the 0.2 ng/µL rhKLK14 mixture in a plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL of 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 (AMC) (Sigma, Catalog # A-9891).
rhKLK14: 0.01 µg
Substrate: 100 µM
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Kallikrein 14 Protein, CF
Kallikrein-like protein 6
kallikrein-related peptidase 14
Human tissue kallikreins refer to a group of secreted serine proteases that are encoded by homologous genes clustering on chromosome 19q13.3-4. As a member of this family, human tissue Kallikrein 14 (hKLK14) is present in many tissues, with high levels in breast, skin, prostate, and brain. The 251 amino acid hKLK14 precursor consists of a signal peptide (residues 1 to 18), a pro peptide (residues 19 to 24) and an active protein (residues 25 to 251) (1). Its enzymatic activity has been shown to be mainly trypsin-like (2). However, its physiological substrates and functions are still unclear. Several studies have suggested that hKLK14 may have clinical utility as a biomarker for cancer of the breast, ovary, and prostate (3, 4). In addition, it may be the initiator of a kallikrein proteolytic cascade responsible for the degradation of the adhesion structures in the stratum corneum (2). The purified, secreted rhKLK14 corresponds to the pro form (residues 19 to 248) with a replacement of the last three residues with a His tag at the C-terminus. When activated by thermolysin, it displays enzymatic activity towards a fluorogenic peptide described above. This activity can be inhibited by recombinant human Serpin A4, E1, and F2 (Catalog # 1669-PI, 1786-PI, and 1470-PI, respectively).
Yousef, G. M. et al. (2001) Cancer Res. 61:3425.
Brattsand, M. et al. (2004) J. Invest. Dermatol. 124:198.
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