MMP-12-M (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for MMP-12-M (Recombinant) on SDS-PAGE or Western blot?

Full-length human MMP-12 (470 aa) has a predicted molecular weight of ~54 kDa, but the expressed construct in REC-MMP12M is produced in HEK293 cells and may represent the catalytic domain and/or propeptide region rather than the full-length protein — check your certificate of analysis for exact sequence boundaries. Mammalian glycosylation from HEK293 expression typically shifts the apparent MW 2–5 kDa above the calculated value on reducing SDS-PAGE. Confirm activation state (pro vs. active form) before interpreting band position; the active catalytic domain runs closer to ~40 kDa.

What processing forms or isoforms of MMP-12 does this recombinant represent, and is it in active or zymogen form?

MMP-12 is synthesized as a ~54 kDa zymogen (including signal peptide and propeptide) and processed to an active ~45 kDa form upon propeptide removal, with further C-terminal truncation yielding an active ~22 kDa catalytic domain. REC-MMP12M is produced by transient HEK293 expression; the exact construct boundaries and activation state — whether zymogen, propeptide-removed active enzyme, or isolated catalytic domain — are specified on the certificate of analysis. If your downstream assay requires fully active enzyme, verify activation status before use and do not assume activity without confirming the construct form.

What substrates does recombinant MMP-12 cleave and what is its substrate preference for activity assays?

MMP-12 is a broad-spectrum elastolytic endopeptidase. It cleaves elastin, gelatin, fibronectin, vitronectin, and several collagens. At the scissile bond, MMP-12 favours aromatic or bulky hydrophobic residues (e.g., Tyr, Phe, Leu) at P1 and leucine at P1′, with small hydrophobics such as alanine tolerated at P1′ as well. For fluorogenic activity assays, FRET-based substrates such as Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 or the generic MMP substrate Mca-PLGL-Dpa-AR are widely used and compatible with REC-MMP12M. Confirm substrate concentration and Km in your specific buffer conditions before calculating specific activity.

What buffer conditions should I use for recombinant MMP-12-M activity assays and does glycerol interfere?

REC-MMP12M is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. The CaCl2 is essential for MMP catalytic domain stability and must be maintained in your assay buffer at 1–5 mM. For fluorogenic assays, dilute into a working buffer of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35 — the detergent prevents surface adsorption at low enzyme concentrations. Glycerol carried over from storage is generally tolerable at <1% final concentration but can quench some fluorophores; keep dilution factor ≥10× when possible.

What starting concentration of recombinant MMP-12 should I use for a fluorogenic activity assay IC50 experiment?

For IC50 determinations against small-molecule or protein inhibitors, a practical starting point is 1–5 nM REC-MMP12M with a fluorogenic substrate (e.g., Mca-PLGL-Dpa-AR) at 10–20 µM (~1× Km). Using enzyme at or below Km allows accurate Ki estimation via the Cheng-Prusoff equation. At concentrations above 10 nM, tight-binding inhibitors (e.g., TIMP-1/TIMP-2) will show stoichiometric rather than hyperbolic inhibition curves, complicating IC50 fits. Always pre-incubate enzyme with inhibitor for 15–30 min at room temperature before adding substrate to allow equilibrium binding.

Can I use MMP-12-M (Recombinant) as a positive control for Western blot with the matched rabbit polyclonal antibody RP-MMP12M?

Yes — REC-MMP12M and antibody RP-MMP12M (see /anti-mmp-12m-rabbit-polyclonal-antibody) are developed in the same laboratory and validated as a matched pair for Western blot positive controls. Loading 20–50 ng of REC-MMP12M per lane on a standard 10–12% SDS-PAGE gel gives a clear, clean band with RP-MMP12M at the expected apparent MW under reducing conditions. This combination is also suitable for validating RP-MMP12M against endogenous MMP-12 in macrophage lysates (e.g., LPS/IFN-γ stimulated THP-1 or primary BMDM), where the recombinant serves as a size and signal reference.

How much recombinant MMP-12-M should I load as a Western blot positive control alongside cell lysate samples?

Load 20–50 ng of REC-MMP12M per lane alongside your cell or tissue lysates. At 20 ng, RP-MMP12M (used at a typical 1:1000–1:2000 dilution) produces a readily detectable band without overwhelming the ECL signal or bleeding into adjacent lanes. If your lysate samples are run at 20–40 µg total protein, a 25 ng recombinant lane provides a proportionate signal reference. Because MMP-12 is a secreted enzyme, include conditioned media concentrate controls where relevant — the recombinant lane confirms antibody and transfer efficiency independently of intracellular expression levels.

How should I store and handle REC-MMP12M to preserve activity after arrival, and what is its shelf life?

Upon receipt, centrifuge briefly and store REC-MMP12M at -20°C in single-use aliquots — the 10% glycerol in the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2) provides cryoprotection. Avoid repeated freeze-thaw cycles; each cycle can measurably reduce specific activity of zinc-dependent endopeptidases. Aliquot to working volumes (typically 5–10 µL) at first thaw. When diluting for assays, use a carrier buffer containing 0.1% BSA to prevent adsorptive losses at concentrations below 10 nM. Stored correctly, activity is stable for ≥12 months at -20°C. Do not store at 4°C long-term.