ADAMTS-15 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAMTS-15 on SDS-PAGE and Western blot?

The full-length human ADAMTS-15 polypeptide is 950 amino acids, giving a theoretical MW of ~106 kDa. Because this recombinant is produced in HEK293 mammalian cells, N-linked glycosylation adds appreciable mass; the apparent MW on reducing SDS-PAGE typically resolves between 120–135 kDa. Run alongside a broad-range marker. Under non-reducing conditions, intramolecular disulfide bonds can shift mobility slightly. If your observed band falls outside this range, confirm with our matched antibody RP-ADAMTS15, which is validated for Western blot detection of the denatured protein.

Is ADAMTS-15 recombinant the full-length protein or a processed/truncated form?

This product represents the secreted, processed form of human ADAMTS-15 (UniProt Q8TE58). The signal peptide (residues 1–23) is cleaved co-translationally in HEK293 cells, and propeptide removal is expected to occur during secretion, consistent with activation of the zinc-dependent catalytic domain. The preparation therefore reflects the mature, extracellular form rather than a truncated catalytic-domain-only fragment. Researchers studying full-domain interactions — particularly those involving the disintegrin-like or thrombospondin motif regions — should find this format suitable without additional processing steps.

What substrate does ADAMTS-15 cleave and how do I set up a versican cleavage activity assay?

ADAMTS-15's principal characterised substrate is versican (VCAN), cleaved at the Glu-1428↓Ala-1429 bond in the GAGβ domain. For a plate-based activity assay, recombinant versican or a synthetic peptide spanning the cleavage site (e.g., DPEAAE↓AQFIT motif used for related aggrecanases) can serve as substrate. Reactions are typically run in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 at 37°C for 2–18 hours. Cleavage products are detected by Western blot using a neo-epitope antibody recognising the AQFIT-terminus, or by fluorescent peptide assay if a validated ADAMTS-15-specific probe is available.

What buffer conditions and calcium concentration does recombinant ADAMTS-15 require for enzymatic activity?

As a zinc-dependent metalloprotease, ADAMTS-15 requires divalent cation support for catalysis. The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — is also suitable as an assay buffer starting point. Maintain at least 1–5 mM CaCl2 in all working dilutions; chelation with EDTA or EGTA will inhibit activity. Avoid reducing agents such as DTT or β-mercaptoethanol above 0.1 mM, as these can disrupt the catalytic zinc coordination and disulfide architecture. Optimal pH is 7.0–7.5; activity drops noticeably below pH 6.5.

What starting concentration of recombinant ADAMTS-15 should I use in an inhibitor screening or IC50 experiment?

For inhibitor screening assays, a typical starting point is 1–5 nM recombinant ADAMTS-15, adjusted empirically so that substrate turnover is linear and within 10–20% conversion over the assay window. This keeps conditions pseudo-first-order and ensures accurate IC50 determination. Dilute from the stock in assay buffer containing 5 mM CaCl2 immediately before use; avoid pre-diluting into glycerol-free buffer for extended periods. Include a broad-spectrum metalloprotease inhibitor (e.g., GM6001 at 10 µM) as a positive inhibition control to confirm zinc-dependence and validate assay signal window.

Can I use ADAMTS-15 recombinant protein as a Western blot positive control for the RP-ADAMTS15 antibody?

Yes — this is one of the primary intended uses of REC-ADAMTS15 alongside RP-ADAMTS15. Both products originate from the same TPB development pipeline and have been validated together. Load 20–50 ng of recombinant ADAMTS-15 per lane on a 4–12% Bis-Tris gel under reducing conditions. RP-ADAMTS15 is validated for Western blot and should detect the ~120–135 kDa glycosylated band. This pairing is particularly useful when optimising RP-ADAMTS15 for detection in cell lysates or tissue extracts where endogenous ADAMTS-15 signal may be low.

How much recombinant ADAMTS-15 should I load as a positive control when validating the RP-ADAMTS15 antibody?

For antibody validation Western blots, 20–50 ng of REC-ADAMTS15 per lane is recommended as a starting range. At 50 ng with RP-ADAMTS15 at a 1:1000 dilution, a clean band at ~120–135 kDa is expected with standard HRP-secondary detection. If you are titrating the primary antibody (e.g., to establish the lowest working dilution for a publication-quality validation panel), run a dilution series of the recombinant (10, 25, 50 ng) in parallel. Refer to the RP-ADAMTS15 product page at /anti-adamts-15-rabbit-polyclonal-antibody for validated dilution ranges and secondary antibody recommendations.

How should I store and handle recombinant ADAMTS-15 to preserve enzymatic activity after receiving it?

Upon receipt, briefly centrifuge the tube, then store immediately at -20°C in the supplied single-use aliquots. The buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — supports stability through multiple freeze-thaw cycles only poorly; repeated cycling measurably reduces specific activity. Thaw each aliquot on ice, use within the same working session, and discard any remainder rather than refreezing. Working dilutions in assay buffer should be prepared fresh and kept on ice. Under recommended storage, activity is expected to be retained for at least 12 months from date of manufacture.