• 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 # ). The IC    ₅₀ value is<10 nM, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com  

• Source

  Mouse myeloma cell line, NS0-derived Arg22-Trp439, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Arg22

• Predicted Molecular Mass

  48 kDa

• SDS-PAGE

  50-56 kDa, reducing conditions

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 # )

• Substrate: Z-Leu-Arg-AMC (Catalog # )

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhCathepsin L to 1 μg/mL in Assay Buffer.

2. Incubate diluted rhCathepsin L on ice for 15 minutes.

3. Dilute incubated rhCathepsin L to 0.25 μg/mL in Assay Buffer.

4. 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.

5. 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.

6. Dilute the reaction mixtures 1/5 in Assay Buffer.

7. Dilute Substrate to 20 μM in Assay Buffer.

8. Load 50 μL of the incubated mixtures in a plate, and start the reaction by adding 50 μL of 20 μM Substrate.

9. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.

10. Derive the 50% inhibition concentration (IC₅₀) value for rhTestican 1 by plotting RFU/min (or specific activity) vs. concentration with 4?PL fitting.

11. The specific activity for rhCathepsin L at each point may be determined using the following formula (if needed):

     *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

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 #  and ) in assays using the fluorogenic peptide substrates (Catalog #  and ).

• References:

1. Bonnet, F. et al. (1992) Biochem. J. 288:565.

2. Alliel, P.M. et al. (1993) Eur. J. Biochem. 214:347.

3. Bonnet, F. et al. (1996) J. Biol. Chem. 271:565.

4. Marr, H.S. and C.-J.S Edgell (2003) Matrix Biol. 22:259.

5. Bocock, J.P. et al. (2003) Eur. J. Biochem. 270:4008.

6. 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