Recombinant Mouse Granzyme B Protein, CF

Catalog #: 1865-SE
1 Citation Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
1865-SE-010

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Recombinant Mouse 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 >2,000 pmol/min/µg as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived mouse Granzyme B protein
Lys17-Ser247, with a C-terminal 10-His tag

Accession #

P04187

N-terminal Sequence
Analysis

Lys17 & Gly19

Structure / Form

Pro form

Predicted Molecular Mass

27 kDa

SDS-PAGE

Multiple bands between 34-38 kDa, reducing conditions

Product Datasheets

1865-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.

1865-SE

Formulation	Supplied as a 0.2 μm filtered solution in HEPES, NaCl and CaCl₂.
Shipping	The product is shipped with polar packs. 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 opening.

Assay Procedure

Materials

Activation Buffer: 50 mM MES, 50 mM NaCl, pH 5.5
Assay Buffer: 50 mM Tris, pH 7.5
Recombinant Mouse Granzyme B (rmGranzyme B) (Catalog # 1865-SE)
Active Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
Substrate: BOC-Ala-Ala-Asp-SBzl (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
96 Well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Dilute rmGranzyme B to 50 µg/mL in Activation Buffer containing 5.5 µg/mL active rmCathepsin C.
Incubate at 37 °C for 4 hours.
Dilute activated rmGranzyme B to 0.4 ng/µL in Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer containing 200 µM DTNB.
Load into a 96 well clear plate 50 µL of the diluted rmGranzyme B. For a Substrate Blank, load 50 µL of the Assay Buffer.
Start the reaction by adding 50 µL of the Substrate/DTNB 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/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:

rmGranzyme B: 0.020 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). Mouse Granzyme B is synthesized as a precursor (247 residues) with a signal and a pro peptide (residues 1-20) and a mature chain (residues 21-247) (4, 5). The recombinant mouse Granzyme B consisting of residues 17 to 247 was expressed and purified. After activation with cathepsin C, it cleaves a thioester substrate described in the Activity Assay Protocol.

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. p. 1549.
Lobe, C.G. et al. (1986) Science 232:858.
Brunet, J.F. et al. (1986) Nature 322:268.

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 Mouse 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

Coxiella burnetii-infected NK cells release infectious bacteria by degranulation
Authors: S Matthiesen, L Zaeck, K Franzke, R Jahnke, C Fricke, M Mauermeir, S Finke, A Lührmann, MR Knittler
Infect. Immun., 2020-10-19;0(0):.
Species: Bacteria
Sample Types: Whole Cells
Applications: Bioassay