Recombinant Mouse Granzyme D Protein, CF Summary
Ile21-Leu252 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||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.
- 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
Background: Granzyme D
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.
- 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.
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