Recombinant Mouse Kallikrein 1 Protein, CF
Recombinant Mouse Kallikrein 1 Protein, CF Summary
|Mouse Kallikrein 5 precursor
Accession # NP_081082
|Mouse Kallikrein 1
Accession # P15947
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 Tris and NaCl.|
|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.
- 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 Mouse Kallikrein 1 (rmKLK1) (Catalog # 7928-SE)
- 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 rmKLK1 to 200 µg/mL in Activation Buffer.
- Dilute Thermolysin to 2 µg/mL in Activation Buffer.
- Combine 20 µL of diluted rmKLK1 with 20 µL of diluted Thermolysin for 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 rmKLK1 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 rmKLK1 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).
- rmKLK1: 0.01 µg
- Substrate: 100 µM
Background: Kallikrein 1
The kallikreins are a family of trypsin‑like serine proteases, many of which are associated with a variety of cancers (1). Kallikrein 1 (KLK1) is also known as tissue kallikrein and urinary kallikrein. An important physiological function of KLK1 is the cleavage of kininogen to release a vasoactive kinin peptide, bradykinin (for rodent KLK1) or lysyl‑bradykinin (for human KLK1) (2, 3). Kinins regulate vasodilation, blood pressure reduction, smooth muscle relaxation and contraction, pain induction and inflammation. Recombinant mouse KLK1 was expressed with a human CD33 signal peptide and a mouse KLK5 pro‑peptide, followed by the mouse KLK1 catalytic domain (residues 25 to 261). The recombinant mouse KLK1 was purified as the latent pro‑form, which is readily activated by treatment with thermolysin.
- Avgeris, M. et al. (2012) Biol. Chem. 393:301.
- Hosoi, K. et al. (1994) J. Biochem. 115:137.
- Kato, H. et al. (1987) J. Biochem. 102:1389.
Product Specific NoticesCoomassie is a registered trademark of Imperial Chemical Industries Ltd.
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