Recombinant Mouse Kallikrein 5 Protein, CF Summary
Product Specifications
Val23-Asn293, with a C-terminal 10‑His tag
Analysis
Product Datasheets
7236-SE
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.
7236-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Glycerol. |
| 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
- Assay Buffer: 50 mM Tris, 0.005% (w/v) Brij-35, pH 8.0
- Recombinant Mouse Kallikrein 5 (rmKLK5) (Catalog # 7236-SE)
- Recombinant Human Kallikrein 5 (rhKLK5) (Catalog # 1108-SE)
- Substrate: Boc-Val-Pro-Arg-AMC (Catalog # ES011)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmKLK5 to 200 µg/mL in Assay Buffer.
- Dilute rhKLK5 to 2.02 µg/mL in Assay Buffer.
- Combine equal volumes of 200 µg/mL rmKLK5 and 2.02 µg/mL rhKLK5. For a control, combine equal volumes of 2.02 µg/mL rhKLK5 and Assay Buffer.
- Incubate at 37 °C for 16 - 20 hours.
- Dilute activated rmKLK5 to 1 µg/mL in Assay Buffer. Perform same dilution on control.
- Dilute Substrate to 800 µM in Assay Buffer.
- In a plate, load 50 µL of 1 µg/mL rmKLK5 and control, and start the reaction by adding 50 µL of 800 µM Substrate to all wells.
- 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 # A9891).
- rmKLK5: 0.050 µg
- Substrate: 400 µM
Background: Kallikrein 5
Tissue Kallikrein 5 (KLK5) is a secreted serine protease with trypsin‑like substrate specificity. KLK5 is highly expressed in the skin, and is also known as stratum corneum tryptic enzyme due to its high expression level in this tissue. KLK5 is thought to play an important role in the process of desquamation (1). KLK5 is a potential biomarker for ovarian and breast cancers (2). It may also be a physiological activator of KLK7 in the stratum corneum (3). Recombinant mouse KLK5 is purified as the proform, and can undergo autoactivation under the appropriate conditions. KLK5 enzymatic activity can be inhibited by the following recombinant human serpins: Serpin‑A1, ‑A5, ‑F2, and ‑G1 (Catalog # 1268-PI, 1266-PI, 1470-PI, and 2488-PI.
- Brattsand M. and T. Egelrud (1999) J. Biol. Chem. 274:30033.
- Yousef G.M. et al. (2003) Cancer Res. 63:3958.
- Kim H. et al. (2001) Br. J. Cancer 84:543.
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