Recombinant Mouse Granzyme B Protein, CF

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

Recombinant Mouse Granzyme B Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a peptide substrate, t-Butyloxycaronyl-Ala-Ala-Asp-ThioBenzyl ester (Boc-AAD-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 >2,000 pmol/min/µg as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Granzyme B protein
  Lys17-Ser247, with a C-terminal 10-His tag
• Accession #: P04187
• N-terminal Sequence: Lys17 & Gly19
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Gzmb
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• 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: Multiple bands between 34-38 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 HEPES, NaCl and CaCl₂.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • 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
  1. Dilute rmGranzyme B to 50 µg/mL in Activation Buffer containing 5.5 µg/mL active rmCathepsin C.
  2. Incubate at 37 °C for 4 hours.
  3. Dilute activated rmGranzyme B to 0.4 ng/µL in Assay Buffer.
  4. Dilute Substrate to 200 µM in Assay Buffer containing 200 µM DTNB.
  5. 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.
  6. Start the reaction by adding 50 µL of the Substrate/DTNB mixture to wells.
  7. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
  8. 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 0.320 cm
  Note: the output of many spectrophotometers is in mOD Per Well:
  • rmGranzyme B: 0.020 µg
  • DTNB: 100 µM
  • Substrate: 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 B Protein, CF

• C11
• CCPI
• CGL1
• CGL-1
• CSPB
• CSP-B
• CTLA1
• CTLA-1
• CTSGL1
• Fragmentin-2
• Granzyme B
• Granzyme-2
• GranzymeB
• GRB
• GrzB
• GZMB
• HLP
• SECT

Background

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.

1. Kam, C.-M. et al. (2000) Biochim. Biophys. Acta 1477:307.
2. Smyth, M.J. et al. (1996) J. Leukoc. Biol. 60:555.
3. Froelich, C.J. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. p. 1549.
4. Lobe, C.G. et al. (1986) Science 232:858.
5. Brunet, J.F. et al. (1986) Nature 322:268.

Publications for Granzyme B (1865-SE)(2)

Publications using 1865-SE

• Wang, CS;Zhong, Q;Wang, ST;Martin-Alonso, C;Neaher, S;Patel, S;Parisi, T;Kirkpatrick, JD;Sequist, LV;Jacks, TE;Bhatia, SN; Multimodal profiling of proinflammatory protease activity identifies caspase-1 as a target for lung cancer interception bioRxiv : the preprint server for biology 2025-07-02 [PMID: 40631193] (FRET, Mouse)
• S Matthiesen, L Zaeck, K Franzke, R Jahnke, C Fricke, M Mauermeir, S Finke, A Lührmann, MR Knittler Coxiella burnetii-infected NK cells release infectious bacteria by degranulation Infect. Immun., 2020-10-19;0(0):. 2020-10-19 [PMID: 32817330] (Bioassay, Bacteria)

Bioinformatics

• Gene Symbol: Gzmb
• Uniprot:
  • P04187()