Recombinant Human Tryptase beta-2 Protein, CF

Catalog #: 3796-SE Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
3796-SE-010

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

Recombinant Human Tryptase beta-2 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 the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The specific activity is >650 pmol/min/µg, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived human Tryptase beta-2/TPSB2 protein
Met1-Pro275, with a C-terminal 10-His tag

Accession #

P20231

N-terminal Sequence
Analysis

Val29

Structure / Form

Pro form

Predicted Molecular Mass

29 kDa

SDS-PAGE

38 kDa, reducing conditions

Product Datasheets

3796-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.

3796-SE

Formulation	Supplied as a 0.2 μm filtered solution in Tris 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, -70 °C as supplied. 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Maturation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
Heparin Incubation Buffer (HIB): 100 µg/mL Heparin (Sigma, Catalog # H3393), 50 mM MES, pH 5.5
Assay Buffer: 50 mM Tris, pH 8.5
Recombinant Human Tryptase beta ‑2/TPSB2 (rhTPSB2) (Catalog # 3796-SE)
Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002), 2 mM stock in DMSO
1, 10 Phenanthroline (Sigma, Catalog # 320056)
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rhTPSB2 to 100 µg/mL in Maturation Buffer containing Thermolysin at a final concentration of 0.1 µg/mL.
Incubate at room temperature for 15 minutes.
Stop Thermolysin activity by adding an equal volume of 10 mM 1, 10 Phenanthroline prepared in HIB.
Dilute the matured rhTPSB2 to 10 µg/mL in HIB.
Incubate for 2 hours at room temperature.
Dilute 10 µg/mL rhTPSB2 to 2.5 µg/mL in HIB.
Dilute 2.5 µg/mL rhTPSB2 to 0.50 µg/mL in Assay Buffer.
Dilute Substrate to 20 µM in Assay Buffer.
Load 50 µL of 0.50 µg/mL rhTPSB2 into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.
Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (RFU/min) x Conversion Factor** (pmol/RFU)
	amount of enzyme (µg)

*Adjusted for Substrate Blank
**Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

rhTPSB2: 0.025 µg
Substrate: 10 µM

Reconstitution Calculator

Background: Tryptase beta-2/TPSB2

Tryptases are trypsin-like serine proteases, and beta tryptases appear to be the main isoenzymes expressed in mast cells (1). They are stored in secretory granules of mast cells, where they form active tetramers with heparin proteoglycan. Because of the unique arrangement of the active sites in the tetramer, which are facing a narrow central pore, beta tryptases are resistant to macromolecule protease inhibitors (2). When mast cells are activated, beta tryptases are released along with other proteins in secretory granules, participating in provoking inflammatory conditions (3). beta tryptases have been implicated as mediators in the pathogenesis of asthma and other allergic disorders.

References

Caughey, G. H. 2004, in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. pp. 1535.
Sommerhoff, C.P. et al. (1999) Proc. Natl. Acad. Sci. USA. 96:10984.
Hallgren, J. and G. Pejler (2006) FEBS J. 273:1871.

Entrez Gene IDs

64499 (Human); 17229 (Mouse); 29268 (Rat)

Alternate Names

EC 3.4.21; EC 3.4.21.59; mast cell tryptase beta II; mast cell tryptase beta III; TPS2; TPS2tryptase beta-2; TPSB2; tryptase beta 2 (gene/pseudogene); tryptase beta 2; tryptase beta II; tryptase beta III; Tryptase beta2; Tryptase beta-2; Tryptase II; tryptase III; tryptase-2; tryptaseB; tryptaseC; Tryptase-C