ADAMTS-18 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant ADAMTS-18 on SDS-PAGE or Western blot?

The full-length human ADAMTS-18 protein (UniProt Q8TE60) encodes 1,221 amino acids with a predicted unprocessed MW of ~136 kDa. However, this HEK293-expressed recombinant carries native-like N-glycosylation, so the apparent MW on SDS-PAGE typically runs higher — expect a diffuse band in the 150–175 kDa range under reducing conditions. Glycosylation heterogeneity can produce slight smearing, which is normal for heavily glycosylated secreted metalloproteinases. Our in-house QC SDS-PAGE confirms >90% purity at this apparent size, so a broad band in that window is expected, not a sign of degradation.

What is the processing state of recombinant ADAMTS-18 — is it the pro-form or mature active enzyme?

This preparation is produced as the active, processed form rather than the latent zymogen. HEK293 expression and secretion allow furin-like pro-protein convertases endogenous to the host cells to cleave the propeptide, releasing the mature metalloproteinase domain. The resulting recombinant retains the catalytic metalloproteinase domain along with its disintegrin-like, thrombospondin motif, cysteine-rich, and spacer domains. If your experiment specifically requires the pro-form or a defined truncation, contact us — but for enzymatic activity assays and most ECM remodelling studies, this active form is the appropriate starting material.

What substrates does recombinant ADAMTS-18 cleave, and how do I set up an activity assay?

Fibronectin is the best-characterised direct substrate: ADAMTS-18 cleaves fibronectin and negatively regulates fibronectin fibrillogenesis (Barbiera et al., 2025, J Biol Chem). For a cleavage activity assay, incubate recombinant ADAMTS-18 (0.5–2 µg) with purified fibronectin (5–20 µg) in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2) at 37°C for 4–18 hours, then resolve by SDS-PAGE and stain or blot for fibronectin fragmentation. Fluorogenic peptide substrates validated for other ADAMTS members (e.g., QELYMSMPD-based peptides) may also be screened, though fibronectin remains the only biochemically validated substrate for ADAMTS-18 to date.

What buffer conditions are optimal for ADAMTS-18 enzymatic activity, and does calcium matter?

As an astacin-clan metalloproteinase, ADAMTS-18 requires Ca²⁺ for both structural stability and catalytic activity. The storage and assay buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 — is formulated to maintain this requirement. Avoid EDTA- or EGTA-containing buffers, as chelation of Zn²⁺ at the active site and Ca²⁺ from structural sites will abolish activity. If your downstream assay includes protease inhibitor cocktails, verify they are EDTA-free. ZnCl2 supplementation (1–10 µM) can be added to assay buffer if activity appears suboptimal, though it is not routinely necessary at the recommended working concentrations.

What starting concentration should I use for recombinant ADAMTS-18 in an ECM remodeling assay?

For fibronectin cleavage and ECM-remodelling experiments, a practical starting range is 0.2–1 µg/mL (roughly 1.3–6.7 nM based on ~150 kDa apparent MW). Titrate upward if cleavage is incomplete after 18 hours at 37°C. For cell-based assays measuring fibronectin fibrillogenesis suppression, published work suggests nanomolar concentrations are sufficient to observe significant inhibition of matrix assembly. Keep working dilutions in the supplied storage buffer to maintain the 5 mM CaCl2 and 10% glycerol that stabilise the enzyme. Avoid diluting below 10 µg/mL in stock before aliquoting, as surface adsorption can cause activity loss at very low concentrations.

Can I use recombinant ADAMTS-18 as a positive control for Western blot with the matched rabbit polyclonal antibody RP-ADAMTS18?

Yes — this is one of the primary design intentions for pairing REC-ADAMTS18 with antibody RP-ADAMTS18. Load 50–200 ng of recombinant ADAMTS-18 per lane under reducing conditions; the antibody should detect the glycosylated band in the 150–175 kDa range. RP-ADAMTS18 is a rabbit polyclonal validated in-house for Western blot, making this recombinant a reliable, identity-confirmed positive control band. This is particularly useful when running lysates from tissues or cell lines where endogenous ADAMTS-18 expression is low or variable. See the RP-ADAMTS18 product page at /anti-adamts-18-rabbit-polyclonal-antibody for recommended antibody dilutions.

How much recombinant ADAMTS-18 should I load per lane for Western blot positive control to avoid oversaturation?

Start with 100 ng per lane; this is generally sufficient for detection with RP-ADAMTS18 at standard dilutions (1:500–1:2,000) without oversaturating the signal. If your film or digital imager is particularly sensitive, titrate down to 25–50 ng. Because ADAMTS-18 runs as a diffuse glycosylated band (~150–175 kDa), signal spread across a wider MW range means you may need slightly more material than you would for a sharp, non-glycosylated recombinant of the same mass. Include this lane alongside your experimental lysates to confirm antibody specificity and confirm correct band identification in complex samples.

How should I store and handle recombinant ADAMTS-18 to preserve enzymatic activity long-term?

Store at -20°C in single-use aliquots immediately upon receipt. The supplied buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol — is formulated for freeze-thaw stability, but repeated cycling will degrade activity. Avoid freeze-thaw more than once. On the day of use, thaw on ice and dilute directly into your assay buffer; do not leave the working solution at room temperature for extended periods. Aliquot volume should match your typical experiment: 5–10 µg aliquots are practical for most activity assays. Under these conditions, stored material retains activity for up to 12 months at -20°C.