Recombinant Human Granzyme B Protein, CF

Catalog #: 2906-SE
1 Citation Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
2906-SE-010

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

Recombinant Human Granzyme B 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 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 #

P10144

N-terminal Sequence
Analysis

Gly19

Structure / Form

Pro form

Predicted Molecular Mass

27 kDa

SDS-PAGE

30-40 kDa, reducing conditions

Product Datasheets

2906-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.

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 (Tocris, Catalog # 5208), 50 mM stock in DMSO
96-well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Activate rhGranzyme B at 100 µg/mL with 10 µg/mL active rmCathepsin C in Activation Buffer.
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).
Dilute activated rhGranzyme B to 0.5 ng/µL in Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer containing 200 µM DTNB.
In a plate load 50 µL of 0.5 ng/µL rhGranzyme B. Include a Substrate Blank containing 50 µL of Assay Buffer.
Start the reaction by adding 50 µL of Substrate mixture to wells.
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 B: 0.025 µg
DTNB: 100 µM
Substrate: 100 µM

Reconstitution Calculator

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

Kam, C.-M. et al. (2000) Biochim. Biophys. Acta 1477:307.
Smyth, M.J. et al. (1996) J. Leukoc. Biol. 60:555.
Froelich, C.J. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al., eds., pp. 1549.
Schmid, J. and C. Weissman (1987) J. Immunol. 139:250.
Caputo, A. et al. (1988) J. Biol. Chem. 263:6363.
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; GranzymeB; GRB; GrzB; GZMB; HLP; SECT

Related Research Areas

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

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