Granzyme B 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, 2). Granzyme B plays an essential role in granule-mediated apoptosis and may have additional roles in rheumatoid arthritis and in bacterial and viral infections (3). It activates various caspases and cleaves proteins such as aggrecan (3). Mouse Granzyme B is synthesized as a precursor (247 residues) with a signal and a pro peptide (residues 1-20) and a mature chain (residues 21-247) (4, 5). The recombinant mouse Granzyme B consisting of residues 17 to 247 was expressed and purified. After activation with cathepsin C, it cleaves a thioester substrate described in the Activity Assay Protocol.
Recombinant Mouse Granzyme B Protein, CF
R&D Systems | Catalog # 1865-SE
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Key Product Details
- R&D Systems NS0-derived Recombinant Mouse Granzyme B Protein (1865-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
Structure / Form
Pro form
Applications
Enzyme Activity
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Product Specifications
Source
Mouse myeloma cell line, NS0-derived mouse Granzyme B protein
Lys17-Ser247, with a C-terminal 10-His tag
Lys17-Ser247, with a C-terminal 10-His tag
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
N-terminal Sequence Analysis
Lys17 & Gly19
Predicted Molecular Mass
27 kDa
SDS-PAGE
Multiple bands between 34-38 kDa, reducing conditions
Activity
Measured by its ability to cleave a peptide substrate, t-Butyloxycaronyl-Ala-Ala-Asp-ThioBenzyl ester (Boc-AAD-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 >2,000 pmol/min/µg as measured under the described conditions.
The specific activity is >2,000 pmol/min/µg as measured under the described conditions.
Formulation, Preparation, and Storage
1865-SE
| Formulation | Supplied as a 0.2 μm filtered solution in HEPES, NaCl and CaCl2. |
| 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.
|
Background: Granzyme B
References
- Kam, C.-M. et al. (2000) Biochim. Biophys. Acta 1477:307.
- Smyth, M.J. et al. (1996) J. Leukoc. Biol. 60:555.
- Froelich, C.J. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. p. 1549.
- Lobe, C.G. et al. (1986) Science 232:858.
- Brunet, J.F. et al. (1986) Nature 322:268.
Alternate Names
CGL-1, CGL1, CSPB, CTLA-1, CTLA1, CTSGL1, Fragmentin-2, Granzyme-2, GRB, GrzB, GZMB, HLP, SECT
Gene Symbol
GZMB
UniProt
Additional Granzyme B Products
Product Documents for Recombinant Mouse Granzyme B Protein, CF
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Mouse Granzyme B Protein, CF
For research use only
Citations for Recombinant Mouse Granzyme B Protein, CF
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Protocols
View specific protocols for Recombinant Mouse Granzyme B Protein, CF (1865-SE):
Materials
- Activation Buffer: 50 mM MES, 50 mM NaCl, pH 5.5
- Assay Buffer: 50 mM Tris, pH 7.5
- Recombinant Mouse Granzyme B (rmGranzyme B) (Catalog # 1865-SE)
- Active Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
- Substrate: BOC-Ala-Ala-Asp-SBzl (SM Biochemicals LLC, Catalog # SMSB05), 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 rmGranzyme B to 50 µg/mL in Activation Buffer containing 5.5 µg/mL active rmCathepsin C.
- Incubate at 37 °C for 4 hours.
- Dilute activated rmGranzyme B to 0.4 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer containing 200 µM DTNB.
- Load into a 96 well clear plate 50 µL of the diluted rmGranzyme B. For a Substrate Blank, load 50 µL of the Assay Buffer.
- Start the reaction by adding 50 µL of the Substrate/DTNB mixture to wells.
- Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
- 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 0.320 cm
Note: the output of many spectrophotometers is in mOD Per Well:
- rmGranzyme B: 0.020 µg
- DTNB: 100 µM
- Substrate: 100 µM
