Recombinant Human Granzyme B Protein, CF
Recombinant Human Granzyme B Protein, CF Summary
Gly19-Tyr247, with a C-terminal 10-His tag
|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.
- 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 (Catalog # EI004), 1 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
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;77(9):2318-2327.
Sample Types: Protein
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