Recombinant Mouse Granzyme D Protein, CF

Catalog #: 1365-SE Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
1365-SE-010

R&D Systems Recombinant Proteins and Enzymes

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Recombinant Mouse Granzyme D 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 >50 pmol/min/µg, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived mouse Granzyme D protein
Ile21-Leu252 with a C-terminal 10-His tag

Accession #

AAA37736

N-terminal Sequence
Analysis

Ile21 & His25

Predicted Molecular Mass

27 kDa

SDS-PAGE

42 kDa, reducing conditions

Product Datasheets

1365-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.

1365-SE

Formulation	Lyophilized from a 0.2 μm filtered solution in HEPES and NaCl.
Reconstitution	Reconstitute at 100 μg/mL in sterile 25 mM HEPES and 200 mM NaCl, pH 7.5.
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 reconstitution.

Assay Procedure

Materials

Assay Buffer: 0.1 M Tris, pH 9.0
Recombinant Mouse Granzyme D (rmGranzyme D) (Catalog # 1365-SE)
Substrate: SUC-Phe-Leu-Phe-SBZL (Bachem, Catalog # M-1740), 10 mM in DMSO
5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130), 10 mM in DMSO
96 well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Preheat plate reader and Assay Buffer at 37 °C.
Dilute rmGranzyme D to 2 ng/µL in preheated Assay Buffer.
Combine equal volumes of Substrate and DTNB for final concentrations of 5 mM of each.
Dilute the Substrate/DTNB mixture to 200 µM in preheated Assay Buffer.
Load 50 µL of the diluted rmGranzyme D into a 96 well clear plate. Include a Substrate Blank containing 50 µL of Assay Buffer.
Start the reaction by adding 50 µL of the diluted Substrate/DTNB mixture to wells.
Read at a wavelength of 405 nm (bottom read) in kinetic mode for 5 minutes.
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 0.32 cm
     Note: the output of many spectrophotometers is in mOD. Per Well:

rmGranzyme D: 0.1 µg
Substrate: 100 µM
DTNB: 100 µM

Reconstitution Calculator

Background: Granzyme D

Granzyme D 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 (1-3). Granzyme D’s functions are largely unknown. However, its selective expression in primary and functional hematopoietic stromal lines correlates with capacity of supporting growth of a pre-T lymphoma clone (4). Together with Granzymes E, F and G, it is regulated through pregnancy and by IL-2 and IL-15 in granulated metrial gland cells (5). The human or rat counterpart of mouse Granzyme D has not been found. Like other granzymes, mouse Granzyme D is not secreted as a zymogen but stored as a fully processed and activated enzyme in the cytoplasmic granules of CTL (1). It is synthesized as a 252 amino acid precursor with an 18 amino acid signal peptide and a 2 amino acid propeptide (1). The mature protein (residues 21-252) is expressed and purified.

References