Recombinant Mouse Tryptase-5/Prss32 Protein, CF Summary
Product Specifications
Ser20-Arg304, 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.
2634-SE
| Formulation | Lyophilized from a 0.2 μm filtered solution in MES and NaCl. |
| Reconstitution | Reconstitute at 200 μg/mL in sterile 50 mM Tris, 10 mM CaCl2, 150 mM NaCl and 0.05% Brij-35 pH 7.5. |
| Shipping | The product is shipped at ambient temperature. 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: (TCNB) 50 mM Tris, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij-35, pH 7.5
- Assay Buffer: 50 mM Tris, 0.25 M NaCl, pH 8.0
- Recombinant Mouse Tryptase‑5/Prss32 (rmTPS-5) (Catalog # 2634-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
- Substrate: Z-Arg-SBzl (SM Biochemicals, Catalog # SMSB01), 10 mM stock in DMSO
- 5,5’Dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma Catalog # D-8130)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rmTPS-5 to 100 µg/mL with Activation Buffer.
- Dilute Thermolysin to 0.4 µg/mL with Activation Buffer.
- Mix equal volumes of 100 µg/mL rmTPS-5 and 0.4 µg/mL Thermolysin for final concentrations of 50 µg/mL and 0.2 µg/mL, respectively.
- Incubate at 37 °C for 1 hour.
- Stop the reaction with 10 mM 1,10 Phenanthroline.
- Dilute activated rmTPS-5 to 4 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer with 200 µM of DTNB.
- Load 50 µL of the 4 ng/µL rmTPS-5 in a clear plate and start the reaction by adding 50 µL of the Substrate/DTNB mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate mix without any rmTPS-5.
- Read at 405 nm (Absorbance), in kinetic mode for 5 minutes.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol |
| ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Using the extinction coefficient 13260 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD Per Well:
- rmTPS-5: 0.20 µg
- DTNB: 100 µM
- Substrate: 100 µM
Reconstitution Calculator
Background: Tryptase-5/Prss32
Tryptase-5 is encoded by Prss32, one of 13 genes on mouse chromosome 17A3.3 that correspond to functional trypsin-like serine proteases (1). The deduced amino acid sequence of mouse Tryptase-5 consists of 331 residues with a signal peptide (residues 1 to 19), a pro region (residue 20 to 53), a catalytic domain (54 to 304), and a C-terminal hydrophobic peptide (residues 305 to 331). The mRNA is expressed in smooth muscle, eye, stomach, uterus and lymph node. Apparently, Prss32 does not seem to have a counterpart in the human genome. The mouse Tryptase-5 (residues 20 to 304) was expressed in the NS0 cells with a foreign signal peptide. The protein was secreted and purified. After being treated by thermolysin, the enzyme is active against a peptide substrate as described in the Activity Assay Protocol.
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