Recombinant Mouse Mast Cell Protease-11/Prss34 Protein, CF

Catalog #: 2857-SE Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
2857-SE-010

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Mouse Mast Cell Protease-11/Prss34 Protein, CF Summary

Product Specifications

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-Arg-ThioBenzyl ester (Z-R-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 >20,000 pmol/min/µg, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived mouse Mast Cell Protease-11/Prss34 protein
Met20-Ser318, with a C-terminal 10-His tag

Accession #

Q80UR4

N-terminal Sequence
Analysis

Met20

Structure / Form

Pro form

Predicted Molecular Mass

34 kDa

SDS-PAGE

48 kDa doublet, reducing conditions

Product Datasheets

2857-SE

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

2857-SE

Formulation	Supplied as a 0.2 μm filtered solution in MES and NaCl.
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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Activation Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, pH 7.5 (TCN)
Assay Buffer: 50 mM Tris, pH 8.0
Recombinant Mouse Mast Cell Protease‑11/Prss34 (rmMCP-11) (Catalog # 2857-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)
96-well Clear Plate (Costar, Catalog # 92592)
Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Dilute rmMCP-11 to 200 µg/mL with Activation Buffer.
Dilute Thermolysin to 0.4 µg/mL with Activation Buffer.
Mix equal volumes of 200 µg/mL rmMCP-11 and 0.4 µg/mL Thermolysin for final concentrations of 50 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 rmMCP-11 to 0.1 ng/µL in Assay Buffer.
Dilute substrate to 200 µM in Assay Buffer with 200 µM of DTNB.
Load 50 µL of the 0.1 ng/µL rmMCP-11 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 rmMCP-11.
Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (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:

rmMCP-11: 0.005 µg
DTNB: 100 µM
Substrate: 100 µM

Reconstitution Calculator

Background: Mast Cell Protease-11/Prss34

Mast Cell Protease-11 (MCP-11) is encoded by Prss34, 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 MCP-11 consists of 318 residues with a signal peptide (residues 1 to 19), a pro region (residue 20 to 34), and a catalytic domain (35 to 318). The mRNA is preferentially expressed in spleen and bone marrow. The mouse MCP-11 (residues 20 to 318) was expressed in the NS0 cells with a foreign signal peptide. After being treated with thermolysin, the purified enzyme is active against a peptide substrate described in the Activity Assay Protocol. Apparently, the human gene corresponding to Prss34 encodes a protein that is not enzymatically active due to a mutation that leads to a premature translation termination codon.

References