ADAMTS-2 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAMTS-2 on SDS-PAGE or Western blot?

The full-length ADAMTS-2 protein (UniProt O95450) encodes 1,211 amino acids with a predicted unprocessed MW of ~134 kDa. Under reducing SDS-PAGE conditions, this recombinant — expressed in HEK293 cells — typically resolves between 150–175 kDa due to N-linked glycosylation added by the mammalian secretory pathway. The exact apparent MW can shift slightly depending on gel percentage; a 6–8% gel is recommended. Under non-reducing conditions, expect a modest upward shift. This glycoform pattern mirrors the native secreted enzyme, which is relevant when using this protein as a positive control alongside antibody RP-ADAMTS2.

What processing form of ADAMTS-2 is present in this recombinant — full-length, propeptide-removed, or mature form?

This recombinant is expressed as the secreted, processed form of ADAMTS-2 from HEK293 cells. The signal peptide (residues 1–29) is removed co-translationally, and the furin-susceptible propeptide is cleaved in the secretory pathway, yielding the mature, catalytically active metalloproteinase domain-containing form. It is not the full 1,211-residue precursor. The mature form retains the ADAMTS ancillary domains — disintegrin-like, thrombospondin type-1, cysteine-rich, and spacer — that are required for substrate recognition and ECM localization. This is the enzymatically relevant species for procollagen N-propeptidase assays.

What substrates does recombinant ADAMTS-2 cleave and how do I set up a procollagen N-propeptidase activity assay?

ADAMTS-2 (EC 3.4.24.14) cleaves the N-terminal propeptides of procollagens I and II but not type III procollagen. For in vitro activity assays, recombinant type I or type II procollagen N-propeptide substrates are used; cleavage is monitored by SDS-PAGE band shift or by fluorogenic peptide substrates spanning the scissile bond. A standard starting point is 10–50 nM recombinant ADAMTS-2 with substrate in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 at 37°C. ADAMTS-2 also cleaves lysyl oxidase (LOX) downstream of the LOX propeptide site (PMID 31152061), which is relevant for LOX-biology experimental designs.

What buffer should I use for ADAMTS-2 recombinant activity assays and are there any inhibitors to avoid?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol — is compatible with direct use in activity assays after dilution. Calcium (≥1 mM free Ca²⁺) is required for catalytic activity; chelators such as EDTA or EGTA will inhibit the enzyme and must be excluded from assay buffers. Likewise, avoid broad-spectrum metalloproteinase inhibitors (e.g., GM6001, 1,10-phenanthroline) unless they are your experimental variable. Zinc supplementation (1–5 µM ZnCl2) can stabilize activity during extended assay incubations. BSA (0.01–0.1%) may be added to dilution buffers to reduce adsorptive loss at low enzyme concentrations.

What starting concentration of recombinant ADAMTS-2 should I use in a dose-response or inhibitor IC50 experiment?

For inhibitor IC50 determinations, a working enzyme concentration of 5–20 nM is a practical starting range — low enough to avoid substrate depletion artifacts yet high enough for reliable signal above background. Confirm the linear range of your activity signal empirically with a titration (0.5–100 nM) before committing to IC50 runs. At concentrations above ~50 nM, substrate depletion can compress the apparent IC50. The 5 mM CaCl2 in the supplied buffer maintains metalloproteinase stability; dilute into assay buffer immediately before use and keep on ice to minimize activity loss prior to the reaction start.

Can I use this recombinant ADAMTS-2 protein as a positive control for Western blot with the matched RP-ADAMTS2 antibody?

Yes — this is a primary use case. REC-ADAMTS2 and RP-ADAMTS2 are produced from the same target sequence in the same lab, making them a guaranteed compatible pair for Western blot positive controls and antibody validation. Load 25–100 ng of REC-ADAMTS2 per lane (a 50 ng load is a reliable starting point on a standard 6–8% gel under reducing conditions). The RP-ADAMTS2 rabbit polyclonal detects a band in the 150–175 kDa region consistent with the glycosylated mature form. This pairing is also appropriate for establishing that your sample prep and transfer conditions are working before committing precious tissue lysates.

How much recombinant ADAMTS-2 should I load for a Western blot positive control lane?

A load of 50 ng per lane is a practical starting point when using the matched RP-ADAMTS2 antibody (SKU: RP-ADAMTS2). If signal is weak at primary antibody dilutions above 1:2000, increase the load to 100 ng. For antibody titration experiments (e.g., determining optimal primary antibody dilution), a two-fold serial dilution series from 200 ng down to 12.5 ng across lanes gives a clean signal-response curve. Run alongside a molecular weight ladder in the 100–250 kDa range. Avoid loading >200 ng if you are running this adjacent to cell lysate lanes, as high recombinant loads can cause band smearing.

How should I store and handle recombinant ADAMTS-2 to preserve enzymatic activity after receipt?

Upon receipt, confirm the protein arrived on dry ice, then transfer immediately to –20°C for long-term storage. The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol) is formulated to maintain stability at –20°C. Aliquots are single-use; repeated freeze-thaw cycles progressively denature metalloproteinases and will reduce activity. For working dilutions, dilute into assay-compatible buffer on ice immediately before use. If aliquots will be used within one week, storage at 4°C is acceptable but not recommended beyond 7 days. Do not dilute to below ~0.1 µg/mL without carrier protein (BSA) to prevent surface adsorption loss.