Recombinant Mouse Granzyme B Protein, CF
Recombinant Mouse Granzyme B Protein, CF Summary
Lys17-Ser247, with a C-terminal 10-His tag
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.
|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.
- 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
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). 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.
- 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.
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