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Recombinant Human Granzyme A Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human Granzyme A Protein, CF Summary

Details of Functionality
Measured by its ability to cleave a colorimetric peptide substrate, N-carbobenzyloxy-Gly-Arg-ThioBenzyl ester (Z-GR-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 >5,000 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Granzyme A protein
Cys26-Val262, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Cys26
Structure / Form
Pro form
Protein/Peptide Type
Recombinant Enzymes
Gene
GZMA
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
28 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 29-33 kDa, reducing conditions
Publications
Read Publication using 2905-SE.

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 reconstitution.
Buffer
Lyophilized from a 0.2 μm filtered solution in MES, NaCl and CaCl2 with Trehalose.
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Reconstitution Instructions
Reconstitute at 100 μg/mL in sterile 25 mM HEPES, 150 mM NaCl and 10 mM CaCl2, pH 7.5.
Assay Procedure
  • Activation Buffer: 0.1 M Tris, pH 9.0
  • Assay Buffer: 50 mM Tris, pH 8.0
  • Recombinant Human Granzyme A (rhGranzyme A) (Catalog # 2905-SE)
  • Lysyl-Endopeptidase (Wako BioProducts, Catalog # 129-02541), stock in deionized water
  • Substrate: Z-Gly-Arg-thiobenzyl ester (MP Biomedicals, Catalog # 03SB00705 or 03SB00710), 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. Activate rhGranzyme A at 50 µg/mL with 0.1 µg/mL Lysyl-Endopeptidase in Activation Buffer.
  2. Incubate at 37 °C for 1 hour.
  3. Dilute activated rhGranzyme A to 0.2 ng/µL in Assay Buffer.
  4. Dilute Substrate to 200 µM in Assay Buffer containing 200 µM of DTNB.
  5. In a plate, load 50 µL of 0.2 ng/µL rhGranzyme A, and start the reaction by adding 50 µL of 200 µM Substrate mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate mixture.
  6. Read absorbance at a wavelength of 405 nm, in kinetic mode for 5 minutes.
  7. 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 0.320 cm
     Note: the output of many spectrophotometers is in mOD. Per Well:
  • rhGranzyme A: 0.01 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 Human Granzyme A Protein, CF

  • CTL Tryptase
  • CTLA3
  • CTLA3Granzyme A (Cytotoxic T-lymphocyte-associated serine esterase-3; Hanukah factorserine protease)
  • Cytotoxic T-lymphocyte proteinase 1
  • EC 3.4.21
  • Fragmentin-1
  • granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3)
  • Granzyme A
  • Granzyme-1
  • GZMA
  • H factor
  • Hanukkah factor
  • HF
  • HFSP
  • HFSPEC 3.4.21.78

Background

Granzyme A 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. Granzyme A is the most abundant protease in CTL and NK cells. It induces caspase‑independent cell death when introduced into target cells by perforin (1). Human granzyme A is synthesized as a precursor (262 residues) with a signal peptide (residues 1‑26), a propeptide (residues 27-28) and a mature chain (residues 29-262 ) (2). The purified recombinant human Granzyme A consists of residues 26 to 262. After being activated by lysyl endopeptidase, it cleaves a thioester substrate as described in Activity Assay Protocol.

  1. Lieberman, J. and Z. Fan (2003) Curr. Opin. Immunol. 15:553.
  2. Gershenfeld, H.K. et al. (1988) Proc. Natl. Acad. Sci. USA 85:1184.

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Publications for Granzyme A (2905-SE)(1)

We have publications tested in 1 application: Enzyme Assay.


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Bioinformatics

Gene Symbol GZMA
Uniprot