Home / Granzyme A / Recombinant Human Granzyme A Protein, CF

Recombinant Human Granzyme A Protein, CF

Catalog #: 2905-SE
1 Citation Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
2905-SE-010
R&D Systems Recombinant Proteins and Enzymes
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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human Granzyme A Protein, CF Summary

Product Specifications

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.
Activity
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 #
P12544
N-terminal Sequence
Analysis
Cys26
Structure / Form
Pro form
Predicted Molecular Mass
28 kDa
SDS-PAGE
Multiple bands between 29-33 kDa, reducing conditions

Product Datasheets

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2905-SE

Product Datasheet
COA
SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

2905-SE

Formulation Lyophilized from a 0.2 μm filtered solution in MES, NaCl and CaCl2 with Trehalose.
Reconstitution Reconstitute at 100 μg/mL in sterile 25 mM HEPES, 150 mM NaCl and 10 mM CaCl2, pH 7.5.
Shipping The product is shipped at ambient temperature. 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, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.

Assay Procedure

Materials
  • 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
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Granzyme A

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.

References
  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.
Entrez Gene IDs
3001 (Human); 14938 (Mouse); 266708 (Rat)
Alternate Names
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

Citation for Recombinant Human Granzyme A 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., 2017-05-01;77(9):2318-2327.
    Applications: Enzyme Assay

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