MMP-14 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant MMP-14 on SDS-PAGE?

The full-length human MMP-14 (UniProt P50281) has a predicted unglycosylated mass of ~65.7 kDa. This HEK293-expressed recombinant form carries native-like N-glycosylation, so the predominant band typically resolves at approximately 68–72 kDa under reducing SDS-PAGE conditions. The mature, prodomain-cleaved catalytic form (roughly residues 112–582 minus the transmembrane anchor in soluble constructs) migrates at ~55–60 kDa. Check lot-specific QC data on the CoA; glycosylation micro-heterogeneity can produce a diffuse band rather than a sharp single species.

What processing form is this recombinant MMP-14 — zymogen or active enzyme?

This product is supplied as the active enzyme, not the latent zymogen. The prodomain has been removed during production in HEK293 cells, which closely mirrors the furin-mediated intracellular activation seen in vivo. It retains the catalytic domain with its zinc-binding HEXXHXXGXXH motif and the hemopexin-like domain, but lacks the transmembrane anchor and cytoplasmic tail — making it suitable for solution-phase activity assays. If your experiment specifically requires the pro-form, it will need to be produced separately.

What substrates does MMP-14 cleave and which fluorogenic peptide substrate works best in vitro?

MMP-14 cleaves fibrillar collagens (types I, II, III), gelatin, laminin-1, fibronectin, and activates proMMP-2 and proMMP-13. For fluorogenic activity assays, the FRET-based substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (MMP substrate I) is well-characterised and broadly used with MMP-14 at substrate concentrations of 5–20 µM. Assays should be conducted in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 buffer, matching the storage buffer exactly. Gelatin-zymography (8% PAGE co-polymerised with 1 mg/mL gelatin) also confirms proteolytic competence after renaturation.

What starting enzyme concentration should I use for a fluorogenic MMP-14 activity assay?

A starting point of 1–5 nM recombinant MMP-14 in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, pH 7.5) is appropriate for most FRET-substrate assays. Use 10 µM Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 as the substrate and monitor fluorescence at Ex/Em 320/400 nm. Include 10 mM EDTA or 1 µM GM6001 as a no-activity control. Titrate enzyme concentration to remain in the linear range; high enzyme loads will deplete substrate rapidly. Equilibrate the protein to room temperature for 10 minutes before adding substrate.

How do buffer conditions affect recombinant MMP-14 activity, and can I substitute the storage buffer?

MMP-14 is a zinc- and calcium-dependent metalloproteinase. The 5 mM CaCl2 in the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is not incidental — calcium stabilises the hemopexin domain and maintains full catalytic activity. Diluting into Ca2+-free buffers or buffers containing EDTA/EGTA will progressively inhibit the enzyme. Keep assay buffers at pH 7.0–7.5; activity drops markedly below pH 6.5. Avoid more than 0.01% Tween-20, which can partially denature the active site at the concentrations sometimes used in ELISA-adjacent setups.

Can I use recombinant MMP-14 as a positive control for Western blot with the matched TPB antibody RP-MMP14?

Yes — this is the intended pairing. REC-MMP14 and RP-MMP14 are produced and validated in the same lab, so epitope coverage is confirmed rather than assumed. Load 20–50 ng of REC-MMP14 per lane for a clean, well-resolved positive control band at ~68–72 kDa (glycosylated form) or ~55–60 kDa (catalytic fragment, lot-dependent). Run alongside your cell lysate lanes. RP-MMP14 is a rabbit polyclonal validated for Western blot; see the antibody product page at /anti-mmp-14-rabbit-polyclonal-antibody for recommended dilutions and secondary antibody pairing.

How much recombinant MMP-14 should I load as a Western blot positive control alongside RP-MMP14?

Load 20–50 ng per lane under reducing conditions. At 50 ng you will get a strong signal that remains within the linear detection range for most HRP-based ECL systems when RP-MMP14 is used at its recommended dilution (see antibody datasheet at /anti-mmp-14-rabbit-polyclonal-antibody). If you are using a high-sensitivity chemiluminescent substrate, start at 20 ng to avoid saturation. Ensure your sample buffer contains β-mercaptoethanol or DTT — reducing conditions are necessary for the antibody to recognise the denatured epitope correctly. Do not boil metalloproteinase samples longer than 5 minutes.

How should I store recombinant MMP-14 and what is the shelf life after thawing?

Store at -20°C in single-use aliquots as supplied. The glycerol in the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) prevents freeze-cracking at -20°C. Repeated freeze-thaw cycles cause measurable loss of catalytic activity — plan aliquot sizes to match a single experiment. Once thawed, keep on ice and use within 4–6 hours; do not re-freeze. Working dilutions in assay buffer should be prepared fresh each day. At -20°C in original aliquots, activity is stable for at least 12 months from the lot manufacture date shown on the CoA.