Recombinant Human Granzyme B Protein, CF

Catalog # Availability Size / Price Qty
2906-SE-010
Product Details
Citations (1)
FAQs
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Recombinant Human Granzyme B Protein, CF Summary

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
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.
Source
Mouse myeloma cell line, NS0-derived human Granzyme B protein
Gly19-Tyr247, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Analysis
Gly19
Structure / Form
Pro form
Predicted Molecular Mass
27 kDa
SDS-PAGE
37 kDa, reducing conditions

Product Datasheets

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.

Assay Procedure

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 (Catalog # EI004), 1 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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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.
Entrez Gene IDs
3002 (Human); 14939 (Mouse)
Alternate Names
C11; CCPI; CGL1; CGL-1; CSPB; CSP-B; CTLA1; CTLA-1; CTSGL1; Fragmentin-2; Granzyme B; Granzyme-2; GRB; GrzB; GZMB; HLP; SECT

Citation for Recombinant Human Granzyme B Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

1 Citation: Showing 1 - 1

  1. Granzyme B PET Imaging as a Predictive Biomarker of Immunotherapy Response
    Authors: BM Larimer, E Wehrenberg, F Dubois, A Mehta, T Kalomeris, K Flaherty, G Boland, U Mahmood
    Cancer Res., 2017;77(9):2318-2327.
    Species: N/A
    Sample Types: Protein
    Applications: EnzAct

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