Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to cleave a colorimetric peptide substrate, N-carbobenzyloxy-Lys-ThioBenzyl ester (Z-Lys-SBzl), in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. The specific activity is >75 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com
Source
Mouse myeloma cell line, NS0-derived Ile29-Phe273, with an C-terminal 10-His tag
Accession #
N-terminal Sequence
AnalysisIle29
Structure / Form
Mature form
Predicted Molecular Mass
28.8 kDa
SDS-PAGE
36 kDa and 37 kDa, reducing conditions
1937-SE |
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Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and CaCl 2. | ||
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.
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Assay Procedure
Materials
Assay Buffer: 50 mM MES, 1 M NaCl, pH 6.5
Recombinant Mouse Tryptase beta -1/MCPT-7 (rmTryptase beta -1) (Catalog # 1937-SE)
Substrate: Benzyloxcarbonyl Lys Thiobenzyl ester (Z-K-SBzl) (Bachem, Catalog # M-1300), 10 mM stock in DMSO
5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130), 10 mM stock in DMSO
96 well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Dilute rmTryptase beta -1 to 10 ng/µL in Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer containing 200 µM of DTNB.
In a plate load 50 µL of 10 ng/µL rmTryptase beta -1, 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 Mixture.
Read in kinetic mode for 15 minutes at an absorbance of 405 nm.
Calculate specific activity using data from 1-15 minutes:
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 0.320 cm
Note: the output of many spectrophotometers is in mOD Per Well:
rmTryptase beta -1: 0.5 µg
DTNB: 100 µM
Substrate: 100 µM
Background: Tryptase beta-1/MCP-7/Mcpt7
Tryptase beta -1 is a serine protease with trypsin-like activity, which is sometimes also referred to as Mast Cell Protease 7 (1). It is stored in the secretory granules of mouse mast cells (2). It exhibits anticoagulant activity due to its ability to degrade fibrinogen in the presence of the diverse array of protease inhibitors in plasma (3). The deduced amino acid sequence for mouse Tryptase beta -1 consists of a signal peptide (residues 1-18), a pro region (residues 19-28) and the mature chain (residue 29‑273). The mature chain was expressed and purified.
References:
McNeil, H.P. et al. (1992) Proc. Natl. Acad. Sci. USA 89:11174.
Matsumoto, R et al. (1995) J. Biol. Chem. 270:19524.
Huang, C. et al. (1997) J. Biol. Chem. 272:31885.
Entrez Gene IDs:
17230 (Mouse)
Alternate Names:
MCP-7; Mcpt7; TPSB1; Tryptase beta1; Tryptase beta-1
