Recombinant Mouse Granzyme G Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Granzyme G Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a peptide substrate, Succinyl-Phe-Leu-Phe-ThioBenzyl ester (Suc-FLF-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 >150 pmol/min/µg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived mouse Granzyme G protein
  Ile21-Leu248, with a C-terminal 10-His tag
  Accession # P13366
• Accession #: P13366
• N-terminal Sequence: Ile21
• Structure / Form: Mature form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Gzmg
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 27 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 35 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 0.1 M Tris, pH 8.0
  • Recombinant Mouse Granzyme G (rmGranzyme G) (Catalog # 1346-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
  1. Dilute rmGranzyme G to 2 ng/µL in Assay Buffer.
  2. Combine equal volumes of Substrate and DTNB for a final concentration of 5 mM of each.
  3. Dilute the Substrate/DTNB mixture to 200 µM in Assay Buffer.
  4. Load 50 µL of the diluted rmGranzyme G into a 96 well clear plate. Include a Substrate Blank containing 50 µL of Assay Buffer.
  5. Start the reaction by adding 50 µL of the diluted Substrate/DTNB mixture to wells.
  6. Read at a wavelength of 405 nm (bottom read) in kinetic mode for 5 minutes.
  7. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 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 G: 0.1 µg
  • Substrate: 100 µM
  • TNB: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Granzyme G Protein, CF

• Ctla7
• Ctla-7
• Granzyme G
• GrzG
• GZMG

Background

Granzyme G 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). Together with Granzymes D, E and F, it is regulated through pregnancy and by IL-2 and IL-15 in granulated metrial gland cells (2). Human or rat counterpart of mouse Granzyme G has not been found. Like other Granzymes, mouse Granzyme G is not secreted as a zymogen but stored as a fully processed and activated enzyme in the cytoplasmic granules of CTL (3). It is synthesized as a 248 amino acid precursor with a 18 amino acid signal peptide and a 2 amino acid propeptide (3, 4). The mature protein (residues 21‑248) is expressed and purified.

1. Smyth, M.J. et al. (1996) J. Leukoc. Biol. 60:555.
2. Allen, M.P. and M. Nilsen-Hamilton (1998) J. Immunol. 161:2772.
3. Jenne, D.E. et al. (1989) Biochemistry 28:7953.
4. Kwon, B.S. et al. (1988) J. Exp. Med. 168:1839.

Bioinformatics

• Gene Symbol: Gzmg
• Uniprot:
  • P13366()