MMP-27-C (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of MMP-27-C (Recombinant) on SDS-PAGE or Western blot?

The full-length human MMP27 open reading frame encodes 513 amino acids, giving a predicted unglycosylated MW of approximately 59 kDa. Because this recombinant is produced in HEK293 cells with native mammalian glycosylation machinery intact, the apparent MW on reducing SDS-PAGE typically runs between 62–68 kDa depending on glycan load. Under non-reducing conditions, intermolecular disulfide-linked species may appear at higher MW. We recommend running 100–200 ng per lane alongside a broad-range marker to resolve the glycoprotein smear characteristic of MMP-family members expressed in mammalian systems.

Is MMP-27-C (Recombinant) the full-length protein or a processed catalytic domain fragment?

This product is produced from the full-length human MMP27 open reading frame (UniProt Q9H306, 513 aa) expressed transiently in HEK293 cells. The signal peptide is cleaved co-translationally, so the secreted/recovered form lacks the N-terminal signal sequence. Both the propeptide and catalytic domains are present, along with the C-terminal hemopexin-like domain. If your experiment specifically requires the isolated catalytic domain or activated (propeptide-removed) form, note the activation state in your assay design and confirm zymogen-vs-active status using the fluorogenic substrate assay described below.

What substrates does MMP-27 cleave and which is recommended for in vitro activity assays?

MMP-27 is reported to cleave fibronectin, laminin, type IV collagen, and gelatins. For routine fluorometric activity assays, a broad-spectrum MMP fluorogenic substrate such as Mca-PLGL-Dpa-AR-NH2 (Ex/Em 328/393 nm) is a practical starting point. Assay in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol — matching the storage buffer exactly — at 37°C. Use 0.1–1 µg recombinant per 100 µL reaction and titrate substrate from 5–100 µM to establish Km. Include 10 mM EDTA or a broad MMP inhibitor (e.g., GM6001) as a negative control.

What starting concentration of MMP-27-C (Recombinant) is recommended for inhibitor IC50 experiments?

For IC50 determination, keep enzyme concentration well below your substrate Km and ideally at or below the Ki of your inhibitor to avoid tight-binding artifacts (Morrison equation territory). A practical starting point is 10–50 nM MMP-27-C in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 37°C. Pre-incubate enzyme with inhibitor for 15–30 min before substrate addition. Confirm linear signal accumulation over the measurement window. EDTA (10 mM) and GM6001 (1 µM final) serve as appropriate positive-inhibition controls given the zinc-dependent catalytic mechanism.

Does the CaCl2 in the storage buffer interfere with downstream assay buffer conditions?

At typical working dilutions the CaCl2 carryover from the storage buffer (5 mM in the stock) is negligible. For example, adding 1 µL of stock to a 100 µL reaction contributes only 0.05 mM CaCl2, well below the 1–5 mM Ca2+ commonly used to support MMP catalytic activity. If your assay is Ca2+-sensitive or uses a chelator, dilute the enzyme into assay buffer first and allow 5 min equilibration on ice. Avoid EGTA/EDTA in the dilution buffer before the final reaction setup, as calcium depletion can destabilize the MMP catalytic domain.

Can I use MMP-27-C (Recombinant) as a positive control for Western blot with the matched RP-MMP27C antibody?

Yes — this is one of the primary reasons REC-MMP27C and RP-MMP27C are co-developed in the same program. Load 50–150 ng of recombinant per lane on a 10% or 12% SDS-PAGE gel under reducing conditions. The RP-MMP27C rabbit polyclonal reliably detects the 62–68 kDa glycoprotein band. This pairing is particularly useful for antibody lot-to-lot validation and for confirming detection sensitivity before moving to low-expressing lysate samples. See the RP-MMP27C product page (/anti-mmp-27c-rabbit-polyclonal-antibody) for recommended primary antibody dilutions.

How much MMP-27-C (Recombinant) should I load for a Western blot positive control lane?

50–150 ng per lane is the recommended range when pairing with RP-MMP27C. Start at 100 ng for initial optimization: this amount reliably produces a detectable band without saturating the signal at standard RP-MMP27C dilutions (typically 1:500–1:2000 for Western blot). If you are running a titration to establish linear detection range for quantitative Western blot work, prepare a 3-point series at 50, 100, and 200 ng. Dilute from the stock into 1× SDS loading buffer immediately before boiling; avoid repeated dilution and freeze-thaw of the working aliquot.

How should I handle, store, and aliquot MMP-27-C (Recombinant) to preserve activity long-term?

Upon receipt, spin briefly at 400 × g, then aliquot into single-use volumes — typically 5–10 µg per tube — in low-binding microcentrifuge tubes. Store at -20°C. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol) is formulated to stabilize metalloproteinase activity through freeze-thaw; however, repeated freeze-thaw cycles progressively reduce specific activity. Do not store at 4°C for more than 48 hours. For activity assays, thaw on ice and use within 2 hours. Purity is >90% by SDS-PAGE and endotoxin is confirmed <0.1 EU/µg by LAL, so no additional clean-up steps are required before use.