MMP-8 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant MMP-8 on SDS-PAGE or Western blot?

The full-length human MMP-8 precursor (467 aa, UniProt P22894) has a predicted MW of ~53 kDa, but the HEK293-expressed recombinant runs at approximately 55–58 kDa under denaturing conditions due to N-linked glycosylation. If you are running the mature, processed form (propeptide removed), expect the band closer to 48–50 kDa. Under reducing conditions (+β-ME or DTT), a single band is expected; non-reducing gels may show higher-MW species from intermolecular disulfide aggregation. Purity is >95% by SDS-PAGE, so minor bands near the main species are glycoform heterogeneity, not contaminating proteins.

Is this recombinant MMP-8 the full-length proenzyme or the processed active form?

REC-MMP8 is supplied as the active enzyme — the propeptide (residues ~19–99) has been processed, exposing the catalytic domain with its HEXXHXXGXXH zinc-binding motif. The hemopexin-like C-terminal domain is retained, which is important for substrate specificity toward fibrillar collagens and for studying TIMP binding interactions. If your experiment requires the latent zymogen form for activation studies, this product is not appropriate; contact us about custom formats. The active form is the predominant species found in PMN granules and is the most physiologically relevant for collagenase activity assays.

What substrates does recombinant MMP-8 cleave, and what fluorogenic peptide should I use for an activity assay?

MMP-8 cleaves fibrillar collagens (types I, II, and III) at the canonical Gly-Ile/Leu bond, initiating triple-helix unwinding. For in vitro fluorogenic activity assays, the widely used substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ (MMP FRET peptide I) is well-validated for MMP-8; excitation at 328 nm, emission at 393 nm. Alternatively, Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys(Dnp)-NH₂ works for broader MMP profiling. A starting enzyme concentration of 1–5 nM in assay buffer with 5 mM CaCl₂ (included in storage buffer) and 0.05% Brij-35 at 37 °C is a practical starting point before titrating for your substrate concentration range.

What buffer conditions work best for MMP-8 collagenase activity assays, and does the storage buffer interfere?

REC-MMP8 is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂. The 5 mM CaCl₂ is intentional — calcium is essential for MMP-8 stability and catalytic activity, so ensure your assay buffer is also supplemented (typically 5–10 mM CaCl₂). Glycerol at 10% can slightly reduce fluorogenic signal; dilute the enzyme at least 1:10 into assay buffer to bring glycerol to ≤1%. Zinc (1 µM ZnCl₂) in assay buffer is advisable for prolonged incubations. Avoid EDTA or EGTA entirely — chelators will inactivate the enzyme. Optimal pH is 7.4–7.6; deviations below 7.0 significantly reduce activity.

How do I determine an appropriate IC50 for an MMP-8 inhibitor using this recombinant, and what enzyme concentration should I use?

For IC50 determinations, keep enzyme concentration well below the inhibitor concentration range you intend to test — typically 1–2 nM REC-MMP8 is appropriate for tight-binding inhibitors (e.g., hydroxamates with Ki in the pM–nM range) to avoid stoichiometric artifacts. Use substrate at or below Km (≈10–20 µM for Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂) to approximate Ki from IC50 via Cheng-Prusoff. Because this protein is HEK293-derived with mammalian glycosylation, inhibitor binding kinetics better reflect those measured against endogenous neutrophil collagenase than bacterially expressed constructs — a meaningful consideration when translating IC50 data to cellular or in vivo models.

Can I use REC-MMP8 as a positive control for Western blot with the matched RP-MMP8 antibody?

Yes — REC-MMP8 and RP-MMP8 are designed as a matched pair for exactly this use case. Load 10–50 ng of REC-MMP8 per lane; 25 ng is a reliable starting point and should produce a clean band at ~55–58 kDa (glycosylated active form) with RP-MMP8 at standard dilutions of 1:500–1:2,000. Because RP-MMP8 is a rabbit polyclonal raised against the human MMP-8 protein, it recognizes multiple epitopes across the mature sequence, making it robust even when your sample is partially degraded. See the RP-MMP8 product page (/anti-mmp-8-rabbit-polyclonal-antibody) for full dilution and blocking conditions.

How much recombinant MMP-8 should I load for a Western blot positive control, and will it look the same as endogenous MMP-8 from cell lysates?

Load 10–50 ng per lane for a clean, unambiguous positive control band. The recombinant runs at ~55–58 kDa due to mammalian glycosylation in HEK293 cells, which closely matches the endogenous neutrophil-derived MMP-8 seen in PMN lysates or conditioned media (~55–75 kDa range depending on glycosylation state and activation status). This alignment makes REC-MMP8 a more informative positive control than bacterial constructs, which typically run lighter (aglycosylated ~48 kDa) and can mislead band-size interpretation. Pair with RP-MMP8 (SKU: RP-MMP8) for a fully validated positive control setup.

How should I handle, aliquot, and store recombinant MMP-8 to preserve activity long-term?

REC-MMP8 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂ and should be stored at −20 °C in single-use aliquots upon receipt. Avoid repeated freeze-thaw cycles — even two additional cycles can reduce collagenase activity by 20–30%. If you anticipate frequent use over 1–2 weeks, keep a working aliquot at 4 °C; activity is stable for up to 7 days under these conditions. Do not dilute in pure water — dilute into assay buffer containing at least 5 mM CaCl₂ and 0.05% Brij-35 to stabilize the enzyme. Carrier protein (0.1% BSA) can be added for very dilute working stocks below 10 nM.