MMP-26 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant MMP-26 on SDS-PAGE or Western blot?

The full-length human MMP-26 protein is 261 amino acids, giving a theoretical MW of ~28 kDa. On reducing SDS-PAGE, this HEK293-expressed recombinant typically resolves at approximately 29–32 kDa, with the slight upward shift attributable to mammalian N-linked glycosylation. Unlike most MMPs, MMP-26 lacks a hemopexin-like domain, so do not expect the higher-MW species (~55–65 kDa) seen with gelatinases. Purity is confirmed at >95% by SDS-PAGE. If running a Western blot positive control alongside antibody RP-MMP26, expect a clean, tight band in that 29–32 kDa window.

Is the recombinant MMP-26 supplied as the zymogen pro-form or the active mature enzyme?

This product (REC-MMP26) is supplied as the active, processed form — the propeptide has been removed during production in HEK293 cells, yielding the mature catalytic domain ready for immediate enzymatic use. The mature catalytic domain spans approximately residues 108–261, and the active-form MW (~18–20 kDa for the catalytic domain alone) is consistent with that expectation. No exogenous APMA activation step is required before activity assays, which simplifies kinetic and inhibitor IC50 experiments and reduces variability introduced by incomplete activation.

What substrates does recombinant MMP-26 cleave and which are best for activity assays?

MMP-26 cleaves type IV collagen, fibronectin, fibrinogen, type I gelatin, beta-casein, and alpha-1 proteinase inhibitor in vitro, and can activate pro-MMP-9. For fluorogenic kinetic assays, a quenched fluorescent peptide substrate such as Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (a generic MMP fluorogenic substrate) works reliably. We have also seen clean activity with Mca-PLGL-Dpa-AR-NH2. Run reactions at 37 °C in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35). Excitation/emission at ~325/393 nm depending on the Mca fluorophore used.

What buffer and assay conditions are recommended for MMP-26 activity and inhibitor IC50 measurements?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol — doubles as a suitable assay base. For IC50 work, supplement with 0.05% Brij-35 to minimize non-specific adsorption of inhibitor or enzyme to plate surfaces, a common source of IC50 variability. CaCl2 at 5 mM is essential for catalytic zinc coordination; omitting it collapses activity. Pre-incubate enzyme with inhibitor for 30–60 min at room temperature before adding substrate. Use 1–5 nM enzyme per well to stay within the linear range of the fluorogenic substrate hydrolysis curve.

What starting concentration of recombinant MMP-26 should I use in a fluorogenic peptide cleavage assay?

For a standard 96-well fluorogenic assay (200 µL volume), start at 1–5 nM REC-MMP26 with substrate at 5–10 µM. At these concentrations you should observe a linear RFU increase for 30–60 min at 37 °C, giving a clean kinetic window for Km and kcat determination. If signal is weak, titrate enzyme up to 10 nM; exceeding ~20 nM often depletes low-concentration substrate too quickly for accurate initial-rate measurements. Always include a no-enzyme background well to subtract intrinsic substrate fluorescence, which can be appreciable with some fluorogenic peptide stocks.

Can I use REC-MMP26 as a positive control for Western blot with the matched RP-MMP26 antibody?

Yes — that pairing is a primary intended use case. Load 20–50 ng of REC-MMP26 per lane alongside your cell lysate samples. Denature under standard reducing conditions (95 °C, 5 min, DTT or β-ME). Probe with RP-MMP26 (rabbit polyclonal, /anti-mmp-26-rabbit-polyclonal-antibody) at manufacturer-recommended dilution (typically 1:500–1:2000). Expect the recombinant to produce a discrete band at ~29–32 kDa — sharper than endogenous MMP-26 in lysates because the recombinant is a single processed form. This lane confirms antibody performance independent of cell-line expression variability.

How much REC-MMP26 do I need per Western blot lane for a reliable positive control band?

20–50 ng per lane is sufficient for a clear, non-saturating signal when using RP-MMP26 at standard dilutions (1:500–1:2000). At 20 ng you retain sensitivity to compare with low-expressing cell lines; at 50 ng the band is robust enough to confirm antibody lot performance at a glance. Avoid loading >100 ng — at high concentrations the recombinant can produce smearing artifacts on nitrocellulose due to glycoprotein stickiness. If quantitative band-intensity comparison with endogenous protein is the goal, titrate both the antibody and the recombinant load in pilot experiments first.

How should I store and handle recombinant MMP-26 to maintain enzymatic activity long term?

REC-MMP26 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol and should be stored at -20 °C in single-use aliquots immediately upon receipt. Repeated freeze-thaw cycles progressively denature the catalytic domain and reduce specific activity — even two extra cycles can cause measurable signal loss in fluorogenic assays. Do not dilute working stocks in plain water; use the supplied buffer or a compatible assay buffer maintaining CaCl2. For same-week experiments, aliquots are stable at 4 °C for up to 5 days. Endotoxin is <0.1 EU/µg by LAL, supporting use in cell-based assays.