Recombinant Human Tryptase alpha/beta-1 Protein, CF
Recombinant Human Tryptase alpha/beta-1 Protein, CF Summary
Product Specifications
Met1-Pro275, 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.
3796-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| 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
- Maturation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Heparin Incubation Buffer (HIB): 100 µg/mL Heparin (Sigma, Catalog # H3393), 50 mM MES, pH 5.5
- Assay Buffer: 50 mM Tris, pH 8.5
- Recombinant Human Tryptase alpha/beta-1 (rhTPSAB1) (Catalog # 3796-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002), 2 mM stock in DMSO
- 1, 10 Phenanthroline (Sigma, Catalog # 320056)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhTPSAB1 to 100 µg/mL in Maturation Buffer containing Thermolysin at a final concentration of 0.1 µg/mL.
- Incubate at room temperature for 15 minutes.
- Stop Thermolysin activity by adding an equal volume of 10 mM 1, 10 Phenanthroline prepared in HIB.
- Dilute the matured rhTPSAB1 to 10 µg/mL in HIB.
- Incubate for 2 hours at room temperature.
- Dilute 10 µg/mL rhTPSAB1 to 2.5 µg/mL in HIB.
- Dilute 2.5 µg/mL rhTPSAB1 to 0.50 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of 0.50 µg/mL rhTPSAB1 into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
- rhTPSAB1: 0.025 µg
- Substrate: 10 µM
Reconstitution Calculator
Background: Tryptase alpha/beta-1
Tryptases are trypsin-like serine proteases, with beta tryptases as the main isoenzymes expressed in mast cells (1). They are stored in secretory granules of mast cells, where they form active tetramers with heparin proteoglycan. Because of the unique arrangement of the active sites in the tetramer, which are facing a narrow central pore, beta tryptases are resistant to macromolecule protease inhibitors (2). When mast cells are activated, beta tryptases are released along with other proteins in secretory granules, participating in provoking inflammatory conditions (3). beta tryptases have been implicated as mediators in the pathogenesis of asthma and other allergic disorders. According to the protein sequence, this product was previously labeled as human Tryptase beta-2 protein upon release. This product has since been renamed as Recombinant Human Tryptase alpha/beta-1 to reflect current sequence alignment per sequence revisions and classification adjustments that have occurred in the NCBI databases.
- Caughey, G. H. 2004, in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. pp. 1535.
- Sommerhoff, C.P. et al. (1999) Proc. Natl. Acad. Sci. USA. 96:10984.
- Hallgren, J. and G. Pejler (2006) FEBS J. 273:1871.
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