Recombinant Mouse Tryptase epsilon/BSSP-4 Protein, CF Summary
Ala33-Ser306, with a C-terminal 10-His tag
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.
|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 and 150 mM NaCl, 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.
- Activation Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl2, pH 7.5 (TCN)
- Assay Buffer: 50 mM Tris, 0.25 M NaCl, pH 8.0
- Recombinant Mouse Tryptase-epsilon /BSSP-4 (rmBSSP-4) (Catalog # 2059-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 in DMSO
- 5,5'Dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma Catalog # D-8130), 10 mM in DMSO
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Reconstitute or dilute rmBSSP-4 to 200 µg/mL with Activation Buffer.
- Dilute Thermolysin to 0.4 µg/mL with Activation Buffer.
- Mix equal volumes of 200 µg/mL rmBSSP-4 and 0.4 µg/mL Thermolysin for final concentrations of 100 µg/mL and 0.2 µg/mL, respectively.
- Incubate at 37 °C for 30 minutes.
- Stop the reaction with 10 mM 1,10 Phenanthroline.
- Dilute activated rmBSSP to 0.4 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer with 200 µM of DTNB.
- Load 50 µL of the 0.4 ng/µL rmBSSP into 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 rmBSSP.
- Read at absorbance wavelength 405 nm 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:
- rmBSSP-4: 0.020 µg
- DTNB: 100 µM
- Substrate: 100 µM
Background: Tryptase epsilon/BSSP-4
Tryptase epsilon, brain specific serine protease 4 (BSSP-4) and brain serine protease 2 (BSP-2) are different names given for the same serine protease that is encoded by the PRSS22 gene (1-3). Initially identified having brain-specific expression, mouse Tryptase epsilon is preferentially expressed in epithelium‑rich tissues such as the lung and eye, which is similar to its human counterpart (3). The mouse protein is synthesized with a signal peptide (amino acid residues 1 to 32), a pro peptide (residues 33 to 49) and a mature chain (residues 50 to 306) corresponding to the serine protease domain. The full-length protein was expressed and the secreted protein purified. The N-terminal sequencing result indicates that the purified protein corresponds to the pro enzyme. After activation with thermolysin, the enzyme has low activity against peptide substrates tested, but high activity against thioester substrates. The thioester activity is inhibited by 2 mM AEBSF (Catalog # EI001), a general serine protease inhibitor, and by recombinant human Serpin A5 (Catalog # 1266-PI).
- Wong, G.W. et al. (2001) J. Biol. Chem. 276:49169.
- Davies, B.J. et al. (1998) J. Biol. Chem. 273:23004.
- Wong, G.W. et al. (2004) J. Biol. Chem. 279:2438.
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