ADAMTS-12 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant ADAMTS-12 on SDS-PAGE or Western blot?

The full-length human ADAMTS-12 has a predicted polypeptide MW of ~178 kDa (1,594 aa; UniProt P58397). Because this recombinant is produced in HEK293 cells, N-linked glycosylation adds appreciable mass — expect the apparent MW on reducing SDS-PAGE to run between 200–230 kDa. Under non-reducing conditions, migration may shift further due to disulfide-stabilized domain architecture. Always run alongside a broad-range ladder (10–250 kDa). Purity is >90% by SDS-PAGE, so the dominant band should be unambiguous at this position.

What processing or isoform state is the recombinant ADAMTS-12 supplied in — prodomain intact or mature form?

REC-ADAMTS12 is supplied as the processed, prodomain-removed mature form, meaning the catalytic domain is accessible and the enzyme is active upon thawing. ADAMTS-12 undergoes furin-mediated cleavage of its prodomain intracellularly; HEK293 expression faithfully recapitulates this step. If your experimental design specifically requires the zymogen/prodomain-intact form for activation studies, this product is not the appropriate choice. For most substrate cleavage, inhibitor binding, or antibody validation workflows, the mature active enzyme is the correct starting material.

What substrates does ADAMTS-12 cleave and which are best for a fluorometric activity assay?

Documented substrates for ADAMTS-12 include aggrecan (at the Glu373–Ala374 aggrecanase site), COMP, alpha-2-macroglobulin, and brain chondroitin sulfate proteoglycans. For a quantitative fluorometric assay, synthetic FRETS-aggrecan peptide substrates or the generic ADAMTS fluorogenic substrate Mca-KPILRGLFRK(Dnp)-NH2 are practical choices and have been used with other ADAMTS family members at Km values typically in the 5–50 µM range. Confirm that 5 mM CaCl2 is present in your assay buffer, as zinc metalloproteinase activity is calcium-dependent and CaCl2 is included in the TPB storage buffer for this reason.

What buffer conditions are optimal for ADAMTS-12 activity assays and are there any inhibitors to avoid?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — is compatible with direct use in activity assays after dilution. For assay buffer, maintain pH 7.4–7.5 and keep CaCl2 at 1–5 mM. Avoid EDTA and EGTA entirely; even low micromolar concentrations chelate the active-site zinc and will abolish activity. Likewise, avoid 1,10-phenanthroline and broad-spectrum hydroxamate inhibitors (e.g., GM6001/ilomastat) in no-inhibitor control wells. DTT above 1 mM can also interfere with disulfide-dependent domain folding.

What starting concentration of recombinant ADAMTS-12 should I use for a substrate cleavage or IC50 inhibitor assay?

For initial substrate cleavage experiments, 1–10 nM enzyme is a practical starting range with synthetic fluorogenic peptide substrates at 10–50 µM. For IC50 determination with small-molecule inhibitors, keep enzyme concentration at or below your estimated Ki to avoid tight-binding artifacts (Cheng-Prusoff correction applies). For COMP or aggrecan cleavage monitored by Western blot or ELISA, 10–50 nM ADAMTS-12 over 2–16 hours at 37°C is commonly reported. Titrate enzyme concentration in pilot experiments rather than assuming published conditions from other ADAMTS paralogs translate directly.

Can I use REC-ADAMTS12 as a positive control for Western blot with the matched RP-ADAMTS12 antibody?

Yes — this is one of the most straightforward uses of the paired products. REC-ADAMTS12 and RP-ADAMTS12 are developed in the same TPB laboratory specifically to be compatible. Load 20–50 ng of recombinant protein per lane on a 6–8% SDS-PAGE gel (reducing conditions); the RP-ADAMTS12 rabbit polyclonal antibody should resolve a clean band at ~200–230 kDa. This positive control is particularly useful when validating RP-ADAMTS12 against cell lysates or tissue samples where endogenous ADAMTS-12 expression may be low. See the antibody product page at /anti-adamts-12-rabbit-polyclonal-antibody for recommended antibody dilutions.

How much recombinant ADAMTS-12 should I load for a Western blot positive control lane?

20–50 ng per lane is sufficient for a strong, clean signal with RP-ADAMTS12 on standard PVDF or nitrocellulose membranes. If you are co-running cell lysates (which will contain far less endogenous ADAMTS-12), load the recombinant in a flanking lane rather than mixing to avoid signal saturation obscuring the lysate bands. Use a 6% or 6–8% gradient gel to resolve the ~200–230 kDa glycoprotein efficiently. For semi-quantitative comparisons, prepare a 2-fold dilution series (50, 25, 12.5, 6.25 ng) to confirm linearity of your detection system before committing to experimental samples.

How should I handle, dilute, and store recombinant ADAMTS-12 to preserve activity over time?

REC-ADAMTS12 is shipped on dry ice and should be stored at -20°C immediately upon receipt. The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) stabilizes the enzyme; do not dialyze away the glycerol or CaCl2. Aliquot upon first thaw into single-use volumes matched to your experiment — repeated freeze-thaw cycles cause measurable activity loss within 3 cycles. For dilutions, use the storage buffer or your assay buffer supplemented with 0.1% BSA (carrier protein) to prevent adsorptive losses at low concentrations (≤10 nM). Working dilutions should be prepared fresh and kept on ice.