Recombinant Human Granzyme B Protein, CF Summary
Product Specifications
Gly19-Tyr247, with a C-terminal 10-His tag
Analysis
Product Datasheets
2906-SE
Carrier Free
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.
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.
2906-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.
|
Assay Procedure
- Activation Buffer: 50 mM MES, 50 mM NaCl, pH 5.5
- Assay Buffer: 50 mM Tris, pH 7.5
- Recombinant Human Granzyme B (rhGranzyme B) (Catalog # 2906-SE)
- Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
- Substrate: t-Butyloxycaronyl-Ala-Ala-Asp-ThioBenzyl ester (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
- (optional) E-64 (Tocris, Catalog # 5208), 50 mM stock in DMSO
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Activate rhGranzyme B at 100 µg/mL with 10 µg/mL active rmCathepsin C in Activation Buffer.
- Incubate at 37 °C for 4 hours. (Optional: use 10 μM E-64 in activation buffer to stop activating enzyme, this is not required under these conditions).
- Dilute activated rhGranzyme B to 0.5 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer containing 200 µM DTNB.
- In a plate load 50 µL of 0.5 ng/µL rhGranzyme B. Include a Substrate Blank containing 50 µL of Assay Buffer.
- Start the reaction by adding 50 µL of Substrate 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/M |
| 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:
- rhGranzyme B: 0.025 µg
- DTNB: 100 µM
- Substrate: 100 µM
Background: Granzyme B
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). Human Granzyme B is synthesized as a precursor (247 residues) with a signal peptide (residues 1-18), a pro peptide (residues 19-20), and a mature chain (residues 21-247) (4-6). The rhGranzyme B consisting of residues 19-247 was expressed and purified. After being activated by active cathepsin C, rhGranzyme B cleaves a thioester substrate described previously (3).
- 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., pp. 1549.
- Schmid, J. and C. Weissman (1987) J. Immunol. 139:250.
- Caputo, A. et al. (1988) J. Biol. Chem. 263:6363.
- Trapani, J.A. et al. (1988) Proc. Natl. Acad. Sci. USA 85:6924.
Citation for Recombinant Human Granzyme B Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Granzyme B PET Imaging as a Predictive Biomarker of Immunotherapy Response
Authors: BM Larimer, E Wehrenberg, F Dubois, A Mehta, T Kalomeris, K Flaherty, G Boland, U Mahmood
Cancer Res., 2017-05-01;77(9):2318-2327.
Applications: Enzyme Assay
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