Recombinant Mouse Tryptase gamma-1/TPSG1 Protein, CF
Recombinant Mouse Tryptase gamma-1/TPSG1 Protein, CF Summary
Product Specifications
His20-Ala275, with a C-terminal 10-His tag
Accession # Q9QUL7
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.
5517-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and CaCl2. |
| 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
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Assay Buffer: 50 mM Tris, pH 8.5
- Recombinant Mouse Tryptase gamma ‑1/TPSG1 (rmTPSG1) (Catalog # 5517-SE)
- Trypsin (Sigma, Catalog # T-1426)
- Recombinant Human Serpin F2/ alpha 2‑Antiplasmin (rhSerpin F2) (Catalog # 1470-PI)
- Fluorogenic Peptide Substrate II: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(Dnp)-NH2 (Catalog # ES002), 2 mM in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmTPSG1 to 200 µg/mL in Activation Buffer.
- Dilute Trypsin to 0.164 µg/mL in Activation Buffer.
- Combine equal volumes of 200 µg/mL rmTPSG1 and 0.164 µg/mL Trypsin in a microfuge tube.
- Incubate at 37 °C for 30 min.
- Dilute rhSerpin F2 to 3.62 µg/mL in Assay Buffer.
- Add rhSerpin F2 to the reaction mixture making a ½ dilution for final concentrations of 1.81 µg/mL rhSerpin F2, 50 µg/mL rmTPSG1, and 0.041 µg/mL Trypsin.
- Incubate for 15 minutes at room temperature. Addition of rhSerpin F2 will stop the activity of Trypsin.
- Dilute rmTPSG1 to 2 ng/µL in Assay Buffer.
- Dilute fluorogenic peptide Substrate to 20 µM in Assay Buffer.
- Load into plate 50 µL of the diluted samples and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 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 Mca-Pro-Leu-OH (Bachem, Catalog # M-1975).
Per Well:- rmTPSG1: 0.100 µg
- Substrate: 10 µM
Reconstitution Calculator
Background: Tryptase gamma-1/TPSG1
Tryptase gamma -1, also known as transmembrane tryptase, is a serine protease synthesized as a preproenzyme with a C-terminal transmembrane anchor (1, 2). Mast cells, mediators of inflammatory and allergic response, are found throughout the body concentrated near blood vessels in connective tissue and the mucous membranes of the respiratory and gastrointestinal tract (3). Upon activation, mast cells release granules that are enriched with neutral serine proteases including tryptases, chymase and cathepsin G (4). The recombinant mouse TPSG1 was expressed as a soluble protein terminated at residue 275, lacking the transmembrane domain, and corresponded to the proenzyme. The proenzyme can be cleaved by trypsin to form the active enzyme. The serine protease activity of trypsin-activated recombinant mouse TPSG1 can be inhibited by ecotin (Catalog # 1328-PI). Typically, >95% protease activity is inhibited by ecotin at approximately a 10:1 molar ratio.
- Wong, G.W. et al. (1999) J. Biol. Chem. 274:30784.
- Caughey, G.H. et al. (2000) J. Immunol. 164:6566.
- Harris, J.L. et al. (2001) J. Biol. Chem. 276:34941.
- Miller, H.R.P. and A.D. Pemberton (2002) Immunology 105:375.
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