ADAMTS-3 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAMTS-3 on SDS-PAGE or Western blot?

The full-length human ADAMTS-3 sequence (1,205 aa, UniProt O15072) predicts an unmodified MW of ~133 kDa. Because this recombinant is produced in HEK293 cells, N-linked glycosylation adds appreciable mass; expect a diffuse band or doublet migrating at approximately 150–170 kDa under reducing SDS-PAGE conditions. Band sharpness improves with PNGase F treatment if you need to confirm the peptide backbone. The prodomain may be partially or fully processed, so a secondary band near 100–110 kDa (mature metalloproteinase-containing fragment) is occasionally observed at higher loads.

Does recombinant ADAMTS-3 cleave type I or type III procollagen, or is it specific to type II?

Under established assay conditions, ADAMTS-3 is selective for the N-propeptide of type II procollagen (pNII collagen substrate) and does not show appreciable activity toward type I or type III procollagen N-propeptides. This substrate selectivity makes it the preferred reagent when you need to isolate type II collagen processing from the broader ADAMTS family activity present in complex lysates. If your experimental model involves type I or III collagen turnover, ADAMTS-2 or ADAMTS-14 are more appropriate choices.

What substrate should I use for an ADAMTS-3 activity assay in vitro?

The best-characterized substrate for ADAMTS-3 activity assays is recombinant type II procollagen N-propeptide (pNII). Fluorogenic FRET peptides spanning the ADAMTS-3 cleavage site in the type II procollagen N-propeptide can also be used to obtain continuous kinetic readouts. Assay buffer should be 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂ — matching the storage buffer to minimize dilution-related activity loss. Zinc chelators (EDTA, 1,10-phenanthroline) and broad-spectrum hydroxamate inhibitors (e.g., GM6001) serve as reliable negative controls to confirm zinc-dependence of the signal.

What starting concentration and buffer conditions should I use when setting up an ADAMTS-3 enzyme activity experiment?

A practical starting point is 1–10 nM recombinant ADAMTS-3 in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂. The enzyme is supplied and stable in this buffer, so diluting directly into matched assay buffer avoids pH or ionic-strength shifts that can denature the metalloproteinase domain. Pre-warm the diluted enzyme at 37 °C for 10 minutes before adding substrate. BSA (0.01–0.1%) can reduce non-specific surface adsorption at low enzyme concentrations. Titrate enzyme concentration against a fixed substrate to confirm you are working in the linear range before running inhibitor or IC₅₀ experiments.

Can I use ADAMTS-3 (Recombinant) as a positive control for Western blot with the matched TPB antibody RP-ADAMTS3?

Yes — this is a primary intended use case. REC-ADAMTS3 and the matched rabbit polyclonal RP-ADAMTS3 (/anti-adamts-3-rabbit-polyclonal-antibody) are validated together for Western blot. Load 50–200 ng of recombinant ADAMTS-3 per lane alongside your experimental samples. The antibody should detect the glycosylated band at ~150–170 kDa. Because the recombinant and antibody originate from the same platform, this pairing is also suitable for confirming antibody lot-to-lot consistency and for establishing that your antigen retrieval or transfer conditions are working before attributing absence of signal to biology.

How much recombinant ADAMTS-3 should I load for a Western blot positive control lane?

Load 50–200 ng in a single lane as your positive control. At 100 ng, a band at ~150–170 kDa is reliably detected with RP-ADAMTS3 at a 1:1,000–1:2,000 primary antibody dilution using standard ECL detection. If you are using a fluorescent secondary system, 50 ng is often sufficient. Avoid loading more than 500 ng per lane — excess glycoprotein can produce smearing that obscures the migration position you intend to benchmark. Run the positive control lane adjacent to your highest-expression sample lane to make direct size comparisons on the same gel straightforward.

How should I store and handle ADAMTS-3 (Recombinant) to maintain enzyme activity long-term?

Store at −20 °C in the supplied single-use aliquots (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂). The 10% glycerol provides cryoprotection; do not dilute it out of the storage aliquot until just before use. Repeated freeze-thaw cycles progressively reduce metalloproteinase activity — even two cycles can cause measurable loss. Once thawed, keep on ice and use within 4–6 hours. If you anticipate multiple uses from one vial, pre-aliquot into single-use volumes upon first receipt. Avoid plastic tubes with high protein-binding capacity; low-bind microcentrifuge tubes are recommended at concentrations below 50 µg/mL.

Is ADAMTS-3 (Recombinant) suitable for antibody validation experiments, and how does it pair with RP-ADAMTS3?

REC-ADAMTS3 is well-suited for antigen-specific antibody validation. Run a dilution series (25, 50, 100, 200 ng) alongside negative lysate (e.g., ADAMTS3-knockout cell lysate or a cell line with no detectable endogenous expression) and probe with RP-ADAMTS3. A single concentration-dependent band confirms specificity; additional bands in the recombinant lane but not the lysate lane are likely glycoform variants rather than off-target reactivity. This approach directly satisfies the recombinant-protein positive control requirement outlined in antibody validation guidelines (e.g., those from the International Working Group for Antibody Validation).