Recombinant Human Testican 1/SPOCK1 Protein, CF

Catalog #: 2327-PI
2 Citations Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
2327-PI-050

R&D Systems Recombinant Proteins and Enzymes

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All Testican 1/SPOCK1 Proteins and Enzymes

Product Details

Citations (2)

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

Recombinant Human Testican 1/SPOCK1 Protein, CF Summary

Product Specifications

Purity

>90%, 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 inhibit active Cathepsin L cleavage of a fluorogenic peptide substrate Z-LR-AMC (Catalog # ES008). The IC₅₀ value is <10 nM, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived human Testican 1/SPOCK1 protein
Arg22-Trp439, with a C-terminal 6-His tag

Accession #

Q08629

N-terminal Sequence
Analysis

Arg22

Predicted Molecular Mass

48 kDa

SDS-PAGE

50-56 kDa, reducing conditions

Product Datasheets

2327-PI

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.

2327-PI

Formulation	Lyophilized from a 0.2 μm filtered solution in PBS.
Reconstitution	Reconstitute at 100 μg/mL in sterile PBS.
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. 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: 50 mM MES, 5 mM DTT, pH 6.0
Recombinant Human Testican 1/SPOCK1 (rhTestican 1) (Catalog # 2327-PI)
Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
Substrate: Z-Leu-Arg-AMC (Catalog # ES008)
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rhCathepsin L to 1 µg/mL in Assay Buffer.
Incubate diluted rhCathepsin L on ice for 15 minutes.
Dilute incubated rhCathepsin L to 0.25 µg/mL in Assay Buffer.
Prepare a curve of rhTestican 1 (MW: 47,800 Da) in Assay Buffer. Make the following serial dilutions: 500, 250, 125, 83.3, 55.6, 37, 24.7, and 12.3 nM.
Combine equal volumes of diluted rhCathepsin L and rhTestican 1 at each concentration of the curve. Include two controls containing equal volumes of Assay buffer and diluted rhCathepsin L without any rhTestican 1.
Incubate mixtures at 37 ˚C for 15 minutes.
Dilute the reaction mixtures 1/5 in Assay Buffer.
Dilute Substrate to 20 µM in Assay Buffer.
Load 50 µL of the incubated mixtures in a plate, and start the reaction by adding 50 µL of 20 µM Substrate.
Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
Derive the 50% inhibition concentration (IC₅₀) value for rhTestican 1 by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
The specific activity for rhCathepsin L at each point may be determined using the following formula (if needed):

Specific Activity (pmol/min/µg) =	Adjusted V_max* (RFU/min) x Conversion Factor** (pmol/RFU)
	amount of enzyme (µg)

*Adjusted for Substrate Blank
**Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

Per Well:

rhCathepsin L: 0.00125 µg
rhTestican 1 curve: 25, 12.5, 6.25, 4.165, 2.78, 1.85, 1.235, and 0.615 nM
Substrate: 10 µM

Reconstitution Calculator

Background: Testican 1/SPOCK1

Testican 1, encoded by the SPOCK1 gene, is a proteoglycan first identified in human seminal plasma (1). The cDNAs from the human testis and mouse brain indicate 95% identity between the deduced amino acid sequences from the two species, predicting a conserved function (2, 3). Human Testican 1 is able to inhibit attachment of Neuro-2a cells and their ability to form neurite extensions (4). At R&D Systems, recombinant human (rh) Testican 1 has also been shown to be able to enhance neurite outgrowth of E18 rat embryonic hippocampal neurons. Testican 1 contains Ca²⁺-binding domain and the C-terminal acidic domain with putative glycosaminoglycan attachment sites (5). In addition, it contains three potential inhibitory domains targeted toward three different classes of proteases, metallo, cysteine and serine proteases. The N-terminal region, which is unique to testicans, is responsible for the inhibition of testican 1 towards MMP-14 (MT1-MMP, a metalloprotease) activation of MMP-2 (6). The thyropin domain may be responsible for the inhibition of testican 1 towards cathepsin L, a cysteine protease (5). The follistatin-like domain with a six cysteine Kazal-like motif may inhibit serine proteases. The purified rhTestican 1 is capable of inhibiting rhMMP-14 and rhCathepsin L (Catalog # 918-MP and 952-CY) in assays using the fluorogenic peptide substrates (Catalog # ES001 and ES008).

References

Bonnet, F. et al. (1992) Biochem. J. 288:565.
Alliel, P.M. et al. (1993) Eur. J. Biochem. 214:347.
Bonnet, F. et al. (1996) J. Biol. Chem. 271:565.
Marr, H.S. and C.-J.S Edgell (2003) Matrix Biol. 22:259.
Bocock, J.P. et al. (2003) Eur. J. Biochem. 270:4008.
Nakada, M. et al. (2001) Cancer Res. 61:8896.

Entrez Gene IDs

6695 (Human)

Alternate Names

FLJ37170; Protein SPOCK; sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1; SPOCK1; SPOCKcwcv and kazal-like domains proteoglycan (testican); Testican 1; TIC1; TICN1

Related Research Areas

Citations for Recombinant Human Testican 1/SPOCK1 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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