Recombinant Human Granzyme H Protein, CF

Catalog #: 1377-SE
2 Citations Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
1377-SE-010

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Citations (2)

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human Granzyme H Protein, CF Summary

Product Specifications

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, Succinyl-Phe-Leu-Phe-ThioBenzyl ester (Suc-FLF-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 H protein
Glu19-Leu246, with a C-terminal 10-His tag
Accession # P20718

Accession #

P20718

N-terminal Sequence
Analysis

Glu19

Structure / Form

Pro form

Predicted Molecular Mass

27 kDa

SDS-PAGE

Multiple bands between 38-40 kDa, reducing conditions

Product Datasheets

1377-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.

1377-SE

Formulation	Supplied as a 0.2 μm filtered solution in HEPES, NaCl and Brij-35.
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, 0.5 M NaCl, pH 9.0
Recombinant Human Granzyme H (rhGranzyme H) (Catalog # 1377-SE)
Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
Substrate: Succinyl-Phe-Leu-Phe ThioBenzyl ester (Suc-FLF-SBzl) (Bachem, Catalog # M-1740), 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 H at 100 µg/mL with 21 µg/mL of rmCathepsin C in Activation Buffer.
Incubate reaction at 37 °C for 2 hours.
Dilute activated rhGranzyme H to 0.4 ng/µL in Assay Buffer.
Dilute Substrate to 200 µM containing 200 µM of DTNB in Assay Buffer.
In a plate load 50 µL of 0.4 ng/µL rhGranzyme H, and start the reaction by adding 50 µL of 200 µM Substrate mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate mixture.
Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (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 H: 0.02 µg
DTNB: 100 µM
Substrate: 100 µM

Reconstitution Calculator

Background: Granzyme H

Granzyme H is a member of the granzyme family of serine proteases found specifically in the cytotoxic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (1, 2). Granzyme H’s functions are largely unknown. The more abundant expression of Granzyme H than Granzyme B in NK cells suggests that Granzyme H may complement the pro-apoptotic function of Granzyme B in this cell type (3). Human Granzyme H shows the highest amino acid identity (71%) to mouse Granzyme C (4). Human Granzyme H is synthesized as a precursor (246 residues) with a signal peptide (residues 1-18), a propeptide (residues 19-20) and a mature chain (residues 21-246) (5-7). The pro-enzyme is expressed and purified. After being activated by active cathepsin C, rhGranzyme H cleaves a thioester substrate, which was described previously (8).

References

Kam, C.-M. et al. (2000) Biochim. Biophys. Acta 1477:307.
Smyth, M.J. et al. (1996) J. Leukoc. Biol. 60:555.
Sedelies, K.A. et al. (2004) J. Biol. Chem. 279:26581.
Sattar, R. et al. (2003) Biochem. Biophys. Res. Comm. 308:726.
Meier, M. et al. (1990) Biochemistry 29:4042.
Haddad, P. et al. (1991) Int. Immunol. 3:57.
Klein, J.L. et al. (1990) Tissue Antigens 35:220.
Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468.

Entrez Gene IDs

2999 (Human)

Alternate Names

Cathepsin G-like 2; CCP-X; CCP-Xcytotoxic T-lymphocyte-associated serine esterase 1; CGL2; CGL-2; CSP-Ccytotoxin serine protease-C; CTLA1; CTSGL2; CTSGL2granzyme H; Cytotoxic serine protease C; Cytotoxic T-lymphocyte proteinase; EC 3.4.21; EC 3.4.21.-; EC 3.4.21.79; granzyme H (cathepsin G-like 2, protein h-CCPX); Granzyme H; GrzH; GZMH

Related Research Areas

Citations for Recombinant Human Granzyme H 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.

2 Citations: Showing 1 - 2
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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
Natural killer cell-derived human granzyme H induces an alternative, caspase-independent cell-death program.
Authors: Fellows E, Gil-Parrado S, Jenne D, Kurschus F
Blood, 2007-04-04;110(2):544-52.
Applications: Enzyme Assay