Granzyme D is a member of the granzyme family of the serine proteases found specifically in the cytotoxic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (1-3). Granzyme D’s functions are largely unknown. However, its selective expression in primary and functional hematopoietic stromal lines correlates with capacity of supporting growth of a pre-T lymphoma clone (4). Together with Granzymes E, F and G, it is regulated through pregnancy and by IL-2 and IL-15 in granulated metrial gland cells (5). The human or rat counterpart of mouse Granzyme D has not been found. Like other granzymes, mouse Granzyme D is not secreted as a zymogen but stored as a fully processed and activated enzyme in the cytoplasmic granules of CTL (1). It is synthesized as a 252 amino acid precursor with an 18 amino acid signal peptide and a 2 amino acid propeptide (1). The mature protein (residues 21-252) is expressed and purified.
Recombinant Mouse Granzyme D Protein, CF
R&D Systems | Catalog # 1365-SE
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Key Product Details
- R&D Systems NS0-derived Recombinant Mouse Granzyme D Protein (1365-SE)
- Quality control testing to verify active proteins with lot specific assays by in-house scientists
- All R&D Systems proteins are covered with a 100% guarantee
Source
NS0
Accession Number
Applications
Enzyme Activity
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Product Specifications
Source
Mouse myeloma cell line, NS0-derived mouse Granzyme D protein
Ile21-Leu252 with a C-terminal 10-His tag
Ile21-Leu252 with a C-terminal 10-His tag
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.
N-terminal Sequence Analysis
Ile21 & His25
Predicted Molecular Mass
27 kDa
SDS-PAGE
42 kDa, reducing conditions
Activity
Measured by its ability to cleave a peptide substrate, Succinyl-Phe-Leu-Phe-ThioBenzyl ester (Suc-FLF-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 >50 pmol/min/µg, as measured under the described conditions.
The specific activity is >50 pmol/min/µg, as measured under the described conditions.
Formulation, Preparation, and Storage
1365-SE
| Formulation | Lyophilized from a 0.2 μm filtered solution in HEPES and NaCl. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile 25 mM HEPES and 200 mM NaCl, pH 7.5. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Calculators
Background: Granzyme D
References
- Jenne, D.E. et al. (1988) Proc. Natl. Acad. Sci. USA 85:4814.
- Kam, C.-M. et al. (2000) Biochim. Biophys. Acta 1477:307.
- Smyth, M.J. et al. (1996) J. Leukoc. Biol. 60:555.
- Yokoi, A. et al. (1999) Biochem. Biophys. Res. Comm. 264:768.
- Allen, M.P. and M. Nilsen-Hamilton (1998) J. Immunol. 161:2772.
Alternate Names
GrzD
Entrez Gene IDs
14941 (Mouse)
Gene Symbol
GZMD
UniProt
Additional Granzyme D Products
Product Documents for Recombinant Mouse Granzyme D Protein, CF
Certificate of Analysis
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Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Mouse Granzyme D Protein, CF
For research use only
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Protocols
View specific protocols for Recombinant Mouse Granzyme D Protein, CF (1365-SE):
Materials
*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:
- Assay Buffer: 0.1 M Tris, pH 9.0
- Recombinant Mouse Granzyme D (rmGranzyme D) (Catalog # 1365-SE)
- Substrate: SUC-Phe-Leu-Phe-SBZL (Bachem, Catalog # M-1740), 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
- Preheat plate reader and Assay Buffer at 37 °C.
- Dilute rmGranzyme D to 2 ng/µL in preheated Assay Buffer.
- Combine equal volumes of Substrate and DTNB for final concentrations of 5 mM of each.
- Dilute the Substrate/DTNB mixture to 200 µM in preheated Assay Buffer.
- Load 50 µL of the diluted rmGranzyme D into a 96 well clear plate. Include a Substrate Blank containing 50 µL of Assay Buffer.
- Start the reaction by adding 50 µL of the diluted Substrate/DTNB mixture to wells.
- Read at a wavelength of 405 nm (bottom read) 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:
- rmGranzyme D: 0.1 µg
- Substrate: 100 µM
- DTNB: 100 µM
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