>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
<1.0 EU per 1 μg of the protein by the LAL method.
48 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
50-56 kDa, reducing conditions
Read Publication using 2327-PI in the following applications:
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 Vmax* (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).
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Testican 1/SPOCK1 Protein, CF
sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1
SPOCKcwcv and kazal-like domains proteoglycan (testican)
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).
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.
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