ADAMTS-4 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAMTS-4 on SDS-PAGE or Western blot?

The full-length human ADAMTS-4 sequence (837 aa, UniProt O75173) predicts a polypeptide backbone of ~93 kDa, but HEK293-derived recombinant typically migrates at 95–100 kDa under denaturing conditions due to N-linked glycosylation. After autocatalytic or furin-mediated prodomain removal, a processed catalytic form (~68 kDa) may also be present; examine your lot-specific SDS-PAGE gel image included with the Certificate of Analysis to confirm which form predominates in your aliquot. Purity is ≥90% by SDS-PAGE.

Which processing or isoform state is the recombinant ADAMTS-4 supplied in — full-length prodomain or processed active form?

This recombinant is produced in HEK293 cells and supplied as an active enzyme. HEK293 co-expresses furin-family proprotein convertases that typically cleave the ADAMTS-4 prodomain at the conserved RXXR motif, yielding a processed, catalytically competent form. The predominant species is therefore the furin-processed enzyme rather than the zymogen. Trace amounts of incompletely processed full-length protein can co-migrate at ~100 kDa. Refer to the CoA for the lot-specific band pattern; the catalytic domain alone is fully sufficient for standard aggrecanase activity assays.

What substrate does ADAMTS-4 cleave and how do I set up a fluorescent activity assay in vitro?

ADAMTS-4 is the principal aggrecanase cleaving aggrecan at the Glu392–Ala393 bond within the interglobular domain. For a quantitative fluorescent assay, use a FRET-based peptide substrate spanning that cleavage site (e.g., Mca-TEGE↓ARGSVI-Dnp) at 10–50 µM in assay buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 0.05% Brij-35. Start with 10–50 ng of recombinant ADAMTS-4 per 100 µL reaction at 37 °C, monitoring fluorescence at Ex/Em 320/405 nm. Purified aggrecan core protein or COMP can substitute for peptide substrates in gel-based cleavage assays.

What buffer conditions are optimal for ADAMTS-4 recombinant activity and are calcium or zinc supplements required?

ADAMTS-4 is a zinc-dependent metalloprotease that additionally requires calcium for full activity and structural stability. The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂ — is compatible with most activity assays and can be used directly. For diluted working stocks, supplement assay buffer with 5 mM CaCl₂. Avoid EDTA or EGTA chelators; these will inactivate the enzyme. Exogenous ZnCl₂ is not required and can be inhibitory above 0.1 mM. Optimal pH range is 7.0–8.0; activity drops sharply below pH 6.5.

What starting concentration of recombinant ADAMTS-4 should I use for an aggrecan cleavage assay and how do I determine linearity?

A reasonable starting point is 5–25 ng of ADAMTS-4 per reaction with 1–5 µg purified aggrecan substrate, incubated 2–16 hours at 37 °C. For FRET peptide assays, 10–20 ng/100 µL typically yields a linear rate over 30–60 minutes. To confirm you are in the linear range, run a time-course at two enzyme concentrations (e.g., 10 ng and 50 ng) and verify that doubling enzyme doubles signal. If running IC₅₀ inhibitor studies, establish the minimum enzyme concentration giving a robust S/B ratio (≥3:1) before titrating test compounds.

Can I use recombinant ADAMTS-4 as a Western blot positive control with the matched TPB antibody RP-ADAMTS4?

Yes — this is a primary use case for pairing REC-ADAMTS4 with RP-ADAMTS4. The rabbit polyclonal antibody (SKU: RP-ADAMTS4) is produced and validated in the same laboratory, so epitope compatibility is confirmed rather than assumed. Load 20–50 ng of recombinant per lane; the processed ~68 kDa and/or full-length ~95–100 kDa bands provide a clean, defined positive control. This is particularly useful when blotting low-expressing cell lysates where an endogenous ADAMTS-4 band may be ambiguous. See the RP-ADAMTS4 product page at /anti-adamts-4-rabbit-polyclonal-antibody for validated dilution ranges.

How much recombinant ADAMTS-4 should I load for Western blot to validate antibody RP-ADAMTS4 specificity?

For antibody specificity validation, 10–50 ng of REC-ADAMTS4 per lane is the recommended range. At 10 ng, a sensitive HRP-based detection system (e.g., ECL Plus, 1–5 min exposure) should yield a detectable band with minimal background. At 50 ng, the band is robust enough for stripped-membrane re-probing or multiplexed fluorescent detection. Run the recombinant alongside a cell lysate known to express ADAMTS-4 (e.g., IL-1β–stimulated chondrocyte or synoviocyte lysate) so band identity in the biological sample can be confirmed by co-migration with the defined molecular weight standard.

How should I store and handle recombinant ADAMTS-4 after receiving it to maintain activity?

Upon receipt, briefly centrifuge the vial, then aliquot into single-use volumes before freezing at −20 °C. Repeated freeze-thaw cycles cause measurable activity loss — even two cycles can reduce recoverable activity by 20–30% for metalloproteinases. Working dilutions should be prepared in storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 10% glycerol) or assay buffer supplemented with 0.1% BSA to reduce surface adsorption at low concentrations. Diluted stocks are stable for up to 8 hours on ice. For long-term storage beyond 6 months, keep at −80 °C.