MMP-27 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant MMP-27 on SDS-PAGE or Western blot?

The full-length MMP-27 polypeptide (UniProt Q9H306) is 513 amino acids, giving a predicted molecular weight of ~58 kDa. On reducing SDS-PAGE, the recombinant protein produced in HEK293 cells typically resolves between 60–65 kDa due to N-linked glycosylation and any retained propeptide sequence. Under non-reducing conditions, the apparent MW may shift slightly. If you are using our matched antibody RP-MMP27 for Western blot, load this recombinant as your positive control lane to confirm the antibody is detecting the correct band in your lysate samples.

Is recombinant MMP-27 supplied as the zymogen or the active form, and how is it processed?

The recombinant MMP-27 supplied here is expressed in HEK293 cells and provided as an active enzyme preparation — not as the intact latent zymogen. HEK293 expression supports the post-translational folding and propeptide processing characteristic of secreted metalloproteinases. That said, MMP-27's activation mechanism remains incompletely characterized in the literature; unlike canonical secreted MMPs, MMP-27 has been reported to localize to the endoplasmic reticulum, and its in vivo activating proteinase has not been definitively identified. Functional activity should be confirmed by substrate cleavage assay before use in quantitative experiments.

What fluorogenic substrate and assay buffer should I use to measure MMP-27 proteolytic activity?

For activity assays, MMP-27 is compatible with the broadly used MMP fluorogenic substrate Mca-PLGL-Dpa-AR-NH2 (or equivalent FRET-based peptide substrates bearing a Pro-Leu-Gly↓Leu cleavage site). Run assays in a buffer consistent with the storage formulation: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, supplemented with 0.05% Brij-35 to reduce non-specific surface adsorption. Zinc-dependent activity requires that no chelating agents (EDTA, EGTA) are present. Include a broad-spectrum MMP inhibitor control (e.g., 1 mM GM6001) to confirm signal specificity.

What starting concentration of recombinant MMP-27 is recommended for a fluorogenic activity assay?

As a starting point, titrate MMP-27 between 5–50 nM in the reaction well alongside a fixed substrate concentration of 10 µM fluorogenic peptide. Because MMP-27-specific catalytic efficiency data (kcat/Km) against defined substrates are not yet well established in the published literature, empirical titration is necessary. Prepare serial dilutions from the stock in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35) immediately before use. Pre-incubate enzyme at 37°C for 15 minutes before adding substrate to allow full equilibration.

What extracellular matrix substrates has MMP-27 been reported to cleave?

MMP-27's specific cleavage targets remain incompletely characterized relative to other MMP family members. Based on domain architecture and available literature, MMP-27 shares substrate overlap with the broader MMP family, including fibronectin, gelatins (denatured collagens), and laminin — substrates degraded by multiple MMPs. Cleavage of fibronectin has been reported in the context of MMP-27 tumor biology studies. Collagens in their native triple-helical form have not been robustly established as direct MMP-27 substrates. Researchers designing substrate cleavage experiments should include appropriate MMP family positive controls (e.g., MMP-2 or MMP-9 for gelatin degradation) alongside MMP-27 for comparison.

Can I use recombinant MMP-27 as a positive control for Western blot with the RP-MMP27 antibody?

Yes — this is the primary validated use case for pairing REC-MMP27 with RP-MMP27. The rabbit polyclonal antibody RP-MMP27 was raised and validated in-house at Triple Point Biologics, and compatibility with this recombinant is confirmed. Load 20–50 ng of REC-MMP27 per lane on a standard 10–12% SDS-PAGE gel under reducing conditions; the antibody should detect a band at approximately 60–65 kDa. This pairing is particularly useful when validating RP-MMP27 against cell lysates where endogenous MMP-27 expression may be low or ambiguous. See the RP-MMP27 product page at /anti-mmp-27-rabbit-polyclonal-antibody for recommended antibody dilutions.

How much recombinant MMP-27 should I load for a Western blot positive control lane?

Load 20–50 ng of REC-MMP27 per lane for a strong, clean positive control band when probing with RP-MMP27. At 20 ng, the band is typically detectable without overloading artifacts; at 50 ng, signal is robust enough to compare alongside low-expressing cell lysate lanes. Run alongside a pre-stained MW ladder to confirm the band resolves at 60–65 kDa. Dilute the stock protein in standard SDS reducing sample buffer; the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) is fully compatible with direct boiling in Laemmli buffer.

How should I store and handle recombinant MMP-27 to preserve activity, and what is its shelf life?

Store REC-MMP27 at -20°C in single-use aliquots upon receipt. The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — is formulated to maintain metalloproteinase stability during freeze-thaw. Repeated freeze-thaw cycles will degrade activity; aliquot to working volumes before the first freeze. Once thawed, keep on ice and use within the same working session. Do not dilute into buffers containing EDTA or other metal chelators, as these will strip the catalytic zinc. Under recommended storage conditions, shelf life is 12 months from date of manufacture.