Recombinant Mouse Testisin/Prss21 Protein, CF Summary
Product Specifications
Leu22-Asn298, with a C-terminal 10-His tag
Analysis
Product Datasheets
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.
6820-SE
| Formulation | Supplied as a 0.2 μm filtered solution in MES, NaCl, CaCl2 and Brij-35. |
| Shipping | The product is shipped with dry ice or equivalent. 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, 150 mM NaCl, 10 mM CaCl2, pH 7.5 (TCN)
- Recombinant Mouse Testisin/Prss21 (rmTestisin) (Catalog # 6820-SE)
- Bacterial Thermolysin (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 rmTestisin to 200 ng/μL in Assay Buffer.
- Dilute Thermolysin to 0.2 μg/mL in Assay Buffer.
- Combine equal volumes of the diluted rmTestisin and diluted Thermolysin and incubate at 37 °C for 30 minutes to activate rmTestisin.
- After incubation, stop reaction by adding an equal volume of 20 mM 1, 10-phenanthroline for a final concentration of 10 mM.
- Dilute activated rmTestisin to 1 ng/μL in Assay Buffer.
- Dilute Substrate to 200 μM in Assay Buffer.
- Load into a plate 50 μL of 1 ng/μL activated rmTestisin, and start the reaction by adding 50 μL of 200 μM Substrate. For a Substrate Blank, load 50 μL of Assay Buffer and 50 μL of 200 μM Substrate.
- Read plate at excitation and emission wavelengths of 380 nm and 460 nm, 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)
- rmTestisin: 0.050 μg
- Substrate: 100 μM
Reconstitution Calculator
Background: Testisin/Prss21
Testisin is a serine protease encoded by the PRSS21 gene. Testisin is similar in structure to tryptases, and is also known as Tryptase‑4 (1). It is predicted to have substrate specificity similar to that of trypsin (2). Testisin is most highly expressed in testicular germ cells, but is also present in eosinophils (3). Testisin is known to be required for spermatogenesis, and deficiencies of the enzyme can result in infertility (4, 5). Testisin is anchored to the cell surface through a C-terminal glycosylphosphatidylinositol anchor (6). Recombinant mouse Testisin is expressed with a truncated C-terminus, resulting in its secretion. The recombinant protein was purified as the zymogen and can undergo autoactivation under the appropriate conditions.
- Wong, G.W. et al. (2001) J. Biol. Chem. 276:20648.
- Hooper, J.D. et al. (2000) Biochim. Biophys. Acta 1492:63.
- Inoue, M. et al. (1998) Biochem. Biophys. Res. Comm. 252:307.
- Netzel-Arnett, S. et al. (2009) Biol. Reprod. 81:921.
- Kawano, N. et al. (2010) Biol. Reprod. 83:359.
- Honda, A. et al. (2002) J. Biol. Chem. 277:16976.
Citation for Recombinant Mouse Testisin/Prss21 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.
1 Citation: Showing 1 - 1
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Intersection of Coagulation and Fibrinolysis by the Glycosylphosphatidylinositol (GPI)-Anchored Serine Protease Testisin
Authors: Buzza, MS;Pawar, NR;Strong, AA;Antalis, TM;
International journal of molecular sciences
Species: N/A
Sample Types: Recombinant Protein
Applications: Bioassay
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