• 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    
  Analysis

  Ile29

• Structure / Form

  Mature form

• Predicted Molecular Mass

  28.8 kDa

• SDS-PAGE

  36 kDa and 37 kDa, reducing conditions

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

1. Dilute rmTryptase beta -1 to 10 ng/μL in Assay Buffer.

2. Dilute Substrate to 200 μM in Assay Buffer containing 200 μM of DTNB.

3. 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.

4. Read in kinetic mode for 15 minutes at an absorbance of 405 nm.

5. Calculate specific activity using data from 1-15 minutes:

     *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:

1. McNeil, H.P. et al. (1992) Proc. Natl. Acad. Sci. USA 89:11174.

2. Matsumoto, R et al. (1995) J. Biol. Chem. 270:19524.

3. 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