ADAMTS-14 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAMTS-14 on SDS-PAGE or Western blot?

The full-length human ADAMTS-14 sequence (UniProt Q8WXS8) encodes 1,223 amino acids, giving a calculated polypeptide mass of ~135 kDa. Because this recombinant is produced in HEK293 cells, N-linked glycosylation adds appreciable mass; expect a diffuse band running at approximately 150–170 kDa under reducing SDS-PAGE conditions. The smearing is characteristic of the multiple glycosylation sites in the TSP repeats and spacer region. If you are blotting with RP-ADAMTS14, this is the band range to gate on for a clean positive control.

Is this recombinant ADAMTS-14 the mature active form or the full-length zymogen?

ADAMTS-14 is naturally synthesised as a latent zymogen with a prodomain that must be removed for catalytic activity. This recombinant is supplied as the active enzyme — the prodomain has been processed during HEK293 secretory pathway handling and/or post-purification activation, yielding a form with exposed catalytic zinc-binding site. The prodomain removal also slightly reduces the apparent SDS-PAGE band compared to unprocessed precursor. Lot-specific activation data are available on the CoA; if your assay requires confirmed zymogen as a baseline control, note that this product is not suitable for that use case.

What substrates can I use to measure ADAMTS-14 N-proteinase activity in a fluorescent assay?

ADAMTS-14 displays procollagen type I N-proteinase (pNPI) activity, cleaving the N-propeptide of fibrillar procollagens at the same scissile bond targeted by ADAMTS-2. For a fluorescent activity assay, a FRET-peptide spanning the ADAMTS-2/14 consensus cleavage site in the pro-α1(I) chain (e.g., Mca-EALQGPQTFGER↓GQAQP-Lys(Dnp)) is the most widely validated substrate. Alternatively, the general metalloproteinase substrate Mca-KPLGL↓DPAKG-Lys(Dnp) can confirm bulk metalloproteinase activity but lacks specificity. For quantitative kinetic work, start with 10–50 µM peptide substrate.

What buffer conditions should I use for ADAMTS-14 activity assays, and is the storage buffer compatible?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂ — is formulated to maintain metalloproteinase stability and can be used directly in activity assays at low dilution. For optimal catalytic throughput, dilute into a working assay buffer of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5–10 mM CaCl₂, 0.05% Brij-35. The calcium is essential for active site integrity; do not substitute with EDTA-containing buffers. Avoid reducing agents such as DTT above 1 mM, which can disrupt the disulfide-stabilised ancillary domains.

What starting concentration of recombinant ADAMTS-14 should I use for an enzyme activity assay?

A working starting point for fluorescent FRET-peptide assays is 1–10 nM recombinant ADAMTS-14 in a 100 µL reaction volume. At this range, initial velocity is linear with enzyme concentration for most peptide substrates at 10–50 µM. For native procollagen substrate assays, titrate upward to 5–50 nM, as the macromolecular substrate has a substantially lower kcat/KM than short peptides. Run a no-enzyme blank and a no-substrate blank in parallel. If inhibitor IC₅₀ determinations are planned, use the lowest enzyme concentration that gives a reliable signal-to-noise ratio (≥5:1 above background).

Can I use recombinant ADAMTS-14 as a positive control for Western blot with the RP-ADAMTS14 antibody?

Yes — this is the primary validated use case for pairing REC-ADAMTS14 with RP-ADAMTS14. The rabbit polyclonal antibody (RP-ADAMTS14) was raised and validated in the same laboratory that produced this recombinant, so epitope recognition against the HEK293-expressed glycoform is confirmed. Load 20–50 ng of REC-ADAMTS14 per lane alongside your cell lysate samples. Expect a band at ~150–170 kDa under reducing conditions. This setup also serves as a molecular weight reference and antibody titration anchor when optimising RP-ADAMTS14 for new cell/tissue lysate matrices.

How much recombinant ADAMTS-14 should I load for a Western blot positive control lane?

For a standard chemiluminescent Western blot using RP-ADAMTS14, load 20–50 ng of REC-ADAMTS14 per lane on a 4–12% Bis-Tris gradient gel. At 20 ng, a clean single band at ~150–170 kDa is detectable with RP-ADAMTS14 at 1:1,000–1:2,000 dilution and an HRP-conjugated anti-rabbit secondary at 1:5,000. If your cell lysate lanes contain low endogenous ADAMTS-14 (e.g., non-fibroblast lines), a 50 ng load provides a more visible reference band without saturating the signal. Dilute the recombinant in 1× SDS sample buffer immediately before loading; avoid repeated dilution-and-freeze cycles.

How should I store and handle recombinant ADAMTS-14 to preserve activity over multiple experiments?

Upon receipt, briefly centrifuge the vial, then aliquot into single-use volumes matching your typical assay requirement — 5–10 µL aliquots are practical for most labs. Store at -20°C; the 10% glycerol in the buffer provides cryoprotection for the metalloproteinase active site. Do not store at 4°C long-term: zinc metalloproteinases are susceptible to autolytic degradation and oxidative inactivation within days at refrigerator temperature. Avoid more than one freeze-thaw cycle per aliquot. Working stocks diluted in assay buffer should be used within 4 hours on ice. Shelf life is 12 months from date of receipt under recommended conditions.