Recombinant Human Kallikrein 12 Protein, CF Summary
Details of Functionality
Measured by its ability to cleave the fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011). The specific activity is > 4,000 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Kallikrein 12 protein Ala18-Asn248, with a C-terminal 10-His tag
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.
Applications/Dilutions
Dilutions
Enzyme Activity
Theoretical MW
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.
SDS-PAGE
37 kDa, reducing conditions
Publications
Read Publications using 3095-SE in the following applications:
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.
Buffer
Supplied as a 0.2 μm filtered solution in MES, NaCl and Glycerol.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Assay Procedure
Activation Buffer: 100 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.05% (w/v) Brij-35, pH 8.0
Assay Buffer: 100 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.05% (w/v) Brij-35, pH 7.5
Recombinant Human Kallikrein 12 (rhKLK12) (Catalog # 3095-SE)
Fluorescent Peptide Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011), 10 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rhKLK12 to 100 µg/mL in Activation Buffer.
Incubate 100 µg/mL rhKLK12 at 37 °C for 24 hours.
Dilute rhKLK12 to 0.2 ng/µL in Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer.
Load into plate 50 µL of 0.2 ng/µL rhKLK12, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing Assay Buffer in place of rhKLK12.
Read plate 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).
Per Well:
rhKLK12: 0.01 µg
Substrate: 100 µM
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Kallikrein 12 Protein, CF
DKFZp686H1078
EC 3.4.21
EC 3.4.21.-
Kallikrein 12
Kallikrein-like protein 5
kallikrein-related peptidase 12
KLK12
KLK-L5
KLKL5kallikrein-12
KLK-L5MGC42603
Background
Human tissue Kallikrein 12, encoded by the KLK12 gene, is a secreted serine protease that belongs to the human tissue kallikrein family. It is present in many tissues, such as salivary gland, stomach and breast. KLK12 displays trypsin-like enzymatic activity. This activity can be inhibited by Serpin F2 (Catalog # 1470-PI) . The physiological functions of KLK12 still remain unclear. Its expression is modulated by steroid hormones and is down‑regulated in breast cancer (2). Human KLK12 has three splice variants resulting from alternative splicing of the 3’ end. The KLK12 produced by the R&D Systems corresponds to the full-length classical form, also known as isoform 2 (1). The amino acid sequence of human KLK12 is 80%, 77%, 71% and 66% to that of bovine, canine, mouse, and rat. The recombinant human KLK12 can be autoactivated under the conditions described in the Activity Assay Protocol. The active enzyme has the N-terminal sequence of I22FNGTECGRNS.
Yousef, G.M. et al. (2000) Genomics. 69:331.
Yousef, G.M. and E.P. Diamandis (2001) Endocrine Rev. 22:184.
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