Recombinant Human Granzyme B Protein, CF

R&D Systems | Catalog # 2906-SE

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human Granzyme B Protein (2906-SE)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

NS0

Accession Number

P10144

Structure / Form

Pro form

Applications

Enzyme Activity
View all Granzyme B Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human Granzyme B protein
Gly19-Tyr247, with a C-terminal 10-His tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Gly19

Predicted Molecular Mass

27 kDa

SDS-PAGE

30-40 kDa, reducing conditions

Activity

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 >1,000 pmol/min/µg, as measured under the described conditions.

Formulation, Preparation, and Storage

2906-SE
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.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

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).

References

  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., pp. 1549.
  4. Schmid, J. and C. Weissman (1987) J. Immunol. 139:250.
  5. Caputo, A. et al. (1988) J. Biol. Chem. 263:6363.
  6. Trapani, J.A. et al. (1988) Proc. Natl. Acad. Sci. USA 85:6924.

Alternate Names

CGL-1, CGL1, CSPB, CTLA-1, CTLA1, CTSGL1, Fragmentin-2, Granzyme-2, GRB, GrzB, GZMB, HLP, SECT

Entrez Gene IDs

3002 (Human); 14939 (Mouse)

Gene Symbol

GZMB

UniProt

P10144

Additional Granzyme B Products

Product Documents for Recombinant Human Granzyme B Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human Granzyme B Protein, CF

For research use only

Citations for Recombinant Human Granzyme B Protein, CF

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Protocols

View specific protocols for Recombinant Human Granzyme B Protein, CF (2906-SE):

Materials
  • 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 (Tocris, Catalog # 5208), 50 mM stock in DMSO
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Activate rhGranzyme B at 100 µg/mL with 10 µg/mL active rmCathepsin C in Activation Buffer.
  2. 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).
  3. Dilute activated rhGranzyme B to 0.5 ng/µL in Assay Buffer.
  4. Dilute Substrate to 200 µM in Assay Buffer containing 200 µM DTNB.
  5. In a plate load 50 µL of 0.5 ng/µL rhGranzyme B.  Include a Substrate Blank containing 50 µL of Assay Buffer.
  6. Start the reaction by adding 50 µL of Substrate 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 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

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