ADAMTS-10 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant ADAMTS-10 on SDS-PAGE or Western blot?

The full-length human ADAMTS-10 protein encodes 1,103 amino acids with a predicted unglycosylated MW of ~124 kDa. Because this recombinant is produced in HEK293 cells, N-linked glycosylation will shift the apparent MW on reducing SDS-PAGE to approximately 130–145 kDa, depending on glycan occupancy. A diffuse or doublet band in that range is normal and does not indicate degradation. If you are running a non-reducing gel, expect the band to migrate differently due to disulfide-bond retention. Our matched antibody RP-ADAMTS10 is validated on Western blot and will recognize this form.

Is the recombinant ADAMTS-10 supplied as the full-length precursor or a processed active form?

The recombinant is expressed in HEK293 cells as the full-length precursor but is secreted with the prodomain removed, yielding a processed, active enzyme form consistent with endogenous ADAMTS-10 maturation. ADAMTS family members undergo furin-like proprotein convertase cleavage at a conserved RXXR motif within the secretory pathway. The resulting active species retains the metalloprotease domain, disintegrin-like domain, and thrombospondin type-1 repeats. If your assay requires the zymogen form, additional engineering would be needed; this product is not that species.

What substrates has recombinant ADAMTS-10 been shown to cleave in activity assays?

ADAMTS-10 has documented proteolytic activity toward versican, particularly the GAG-β domain, and has been reported to cleave fibrillin-1 in the extracellular matrix. For in vitro fluorometric activity assays, a generic ADAMTS fluorogenic substrate such as Mca-RPKPVE-Nva-WRK(Dnp)-NH2 (derived from aggrecan) can provide a baseline readout, though cleavage efficiency is lower than for aggrecanases ADAMTS-4/5. When setting up substrate assays, pre-equilibrate the enzyme in assay buffer at 37 °C for 15 minutes before addition of substrate to allow full activation.

What buffer conditions are recommended for ADAMTS-10 activity assays and does the storage buffer interfere?

The storage buffer is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. For activity assays, dilute the enzyme at least 1:10 into your assay buffer — typically 50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35 — so that residual glycerol drops below 1% and does not inhibit enzyme kinetics. Zinc-dependent metalloproteases are sensitive to chelating agents; ensure your buffers are free of EDTA or EGTA. Include 1 mM PMSF or use a serine protease-free background if your substrate preparation is not ultrapure.

What starting concentration of recombinant ADAMTS-10 should I use for an in vitro cleavage or activity assay?

A practical starting point is 50–200 nM enzyme in a final assay volume of 50–100 µL, which is consistent with activity conditions reported for related ADAMTS family members in the literature. For substrate titration experiments, keep the enzyme concentration fixed at ~100 nM and vary substrate from 5 µM to 500 µM to establish Km. Because ADAMTS-10 has lower intrinsic aggrecanase activity compared to ADAMTS-4/5, erring toward the higher end of the enzyme concentration range (150–200 nM) is advisable for fluorogenic substrate assays to achieve a reliable signal-to-background ratio.

Can I use recombinant ADAMTS-10 as a positive control for Western blot with the RP-ADAMTS10 antibody?

Yes — this is one of the primary use cases. ADAMTS-10 recombinant (REC-ADAMTS10) and the matched rabbit polyclonal RP-ADAMTS10 are developed and validated in the same laboratory, so compatibility for Western blot is confirmed, not predicted. Load 50–100 ng of recombinant per lane on a 4–12% Bis-Tris gel under reducing conditions; you should see a clean band at approximately 130–145 kDa. This lane serves simultaneously as a size marker for the processed form and validates antibody performance in your specific lysate preparation or ECL detection system. See the RP-ADAMTS10 product page for recommended antibody dilution ranges.

How much recombinant ADAMTS-10 should I load for a Western blot positive control lane?

Load 50–100 ng per lane for standard chemiluminescence detection with RP-ADAMTS10. At 50 ng you will typically see a strong, clean band; if your ECL system is highly sensitive (e.g., digital imaging with femtogram detection), 25 ng may suffice and will reduce background halo around the band. Avoid loading more than 200 ng — at high concentrations, glycoprotein smearing can obscure band sharpness. Run alongside 20–40 µg of cell or tissue lysate so you can directly compare endogenous signal intensity against the recombinant standard in the same exposure.

How should I store and handle recombinant ADAMTS-10 to preserve activity, and what is the shelf life?

Store at -20 °C in the single-use aliquots provided. The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) stabilizes the enzyme during freezing; do not substitute or dilute before storage. Avoid repeated freeze-thaw cycles — even two additional cycles can reduce metalloprotease activity by 20–30% based on standard QC measurements for this enzyme class. Once thawed, keep on ice and use within 4–6 hours. Shelf life is 12 months from date of manufacture at -20 °C, unopened. If long-term diluted working stocks are needed, add BSA to 0.1% as a carrier.