MMP-18 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant MMP-18 on SDS-PAGE or Western blot?

The full-length human MMP-18 sequence encodes 469 amino acids with a predicted unglycosylated molecular weight of approximately 54 kDa. Because this recombinant is produced in HEK293 cells, N-linked glycosylation shifts the apparent MW to roughly 57–60 kDa under denaturing, reduced SDS-PAGE conditions. If you are running the active, propeptide-cleaved form, expect a band near 47–50 kDa. Always include a reducing agent in sample buffer; the hemopexin-like domain contains disulfide bonds that will retard migration under non-reducing conditions and can produce anomalous high-MW smearing.

Does recombinant MMP-18 include the propeptide, and which processing form is supplied?

The recombinant MMP-18 (REC-MMP18) is supplied as the active enzyme with the signal peptide and prodomain removed — equivalent to the mature secreted collagenase form found in conditioned medium after autocatalytic or furin-mediated activation. The catalytic domain and C-terminal hemopexin-like domain are both present. If your experiment requires the zymogen (full pro-form) for activation kinetics studies, this product is not the appropriate format. The active enzyme is suitable for direct collagen-cleavage assays or as a Western blot band-size standard without a separate APMA activation step.

What collagen substrates and cleavage sites does recombinant MMP-18 act on?

MMP-18 is a classical collagenase that cleaves fibrillar type I, II, and III collagens at a single Gly~Ile/Leu scissile bond within the triple-helical domain, generating characteristic three-quarter and one-quarter fragments. Documented activity also extends to type VII and type X collagen. For fluorometric activity assays, use a collagen-mimetic FRET substrate such as Mca-PLGL-Dpa-AR-NH2 (MMP collagenase consensus peptide); activity is detectable at enzyme concentrations as low as 1–5 nM with this substrate under standard assay conditions.

What assay buffer should I use for MMP-18 collagenase activity assays?

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. For fluorometric FRET substrate assays, dilute REC-MMP18 into assay buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, and 0.05% Brij-35 to prevent surface adsorption at low enzyme concentrations. Maintain CaCl₂ at ≥1 mM; calcium is required for structural stability of the zinc-containing catalytic domain. Avoid EDTA or EGTA; they chelate zinc and calcium and will abolish activity within minutes.

What starting concentration of recombinant MMP-18 is recommended for a peptide substrate activity assay?

A good starting point is 5–20 nM REC-MMP18 with 5–20 µM FRET peptide substrate (e.g., Mca-PLGL-Dpa-AR-NH2) in a 100 µL reaction at 37°C. Monitor fluorescence at Ex/Em 320/405 nm and confirm linearity over 30–60 minutes before calculating specific activity. For IC50 determination of MMP inhibitors, use 5 nM enzyme to remain well below substrate Km and satisfy the Morrison tight-binding correction threshold. At concentrations above 50 nM, auto-inhibition or substrate depletion can compress the dynamic range of inhibitor dose-response curves.

Can recombinant MMP-18 be used as a positive control for Western blot with the RP-MMP18 antibody?

Yes — REC-MMP18 and the matched rabbit polyclonal antibody RP-MMP18 (/anti-mmp-18-rabbit-polyclonal-antibody) are validated together from the same laboratory, specifically for this purpose. Load 10–50 ng of REC-MMP18 per lane alongside your cell lysate samples; the antibody detects the active-form band at ~57–60 kDa (HEK293-glycosylated) and should produce a clean signal at standard WB dilutions. Because both reagents are from the same source lot validation, this pairing eliminates the ambiguity of cross-reactivity with other MMP family members that frequently complicates MMP-18 Western blot interpretation.

How much recombinant MMP-18 should I load as a positive control on a Western blot, and what band should I expect?

Load 20–50 ng of REC-MMP18 per lane for a strong, reproducible positive control band. On a 10% or 4–12% gradient SDS-PAGE gel under reducing conditions, expect the primary band at 57–60 kDa (glycosylated active form). A faint secondary band at ~47–50 kDa may appear if minor propeptide processing occurs during storage — this is not a contamination artifact. Use with RP-MMP18 at the manufacturer's recommended WB dilution. If signal is weak, increasing REC-MMP18 to 50 ng is more reliable than increasing antibody concentration, which raises background.

What are the storage conditions and shelf life for recombinant MMP-18, and how should I handle it after thawing?

Store REC-MMP18 at -20°C in single-use aliquots in its supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 10% glycerol). Shelf life is 12 months from receipt under these conditions. After thawing, keep on ice and use within 4–6 hours; repeated freeze-thaw cycles degrade collagenase activity measurably by the second cycle. Do not dilute into water alone — always maintain CaCl₂ and at least 0.1 mg/mL carrier protein (BSA) if working below 10 nM to prevent adsorptive loss to tube walls. Purity is >90–95% by SDS-PAGE; endotoxin is <0.1 EU/µg by LAL.