Recombinant Human Granzyme A Protein, CF Summary
Cys26-Val262, with a C-terminal 10-His tag
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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.
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.
|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.
- 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
- Activate rhGranzyme A at 50 µg/mL with 0.1 µg/mL Lysyl-Endopeptidase in Activation Buffer.
- Incubate at 37 °C for 1 hour.
- Dilute activated rhGranzyme A to 0.2 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer containing 200 µM of DTNB.
- 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.
- Read absorbance at a wavelength of 405 nm, in kinetic mode for 5 minutes.
- 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
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.
- Lieberman, J. and Z. Fan (2003) Curr. Opin. Immunol. 15:553.
- Gershenfeld, H.K. et al. (1988) Proc. Natl. Acad. Sci. USA 85:1184.
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
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.
Sample Types: Protein
Applications: Enzyme Assay
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