MMP-11 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant MMP-11 on SDS-PAGE or Western blot?

The mature, furin-processed form of MMP-11 runs at approximately 44–46 kDa under reducing SDS-PAGE conditions. Because this recombinant is produced in HEK293 cells with native-like glycosylation, the observed band may migrate slightly higher than the calculated molecular weight of the 488-amino-acid sequence (~54 kDa with propeptide) depending on glycan load. If you are seeing a band near 60 kDa, this likely reflects incomplete propeptide removal or glycosylation heterogeneity — both are expected in a subset of HEK293-expressed MMP-11 preparations.

Is recombinant MMP-11 supplied as the zymogen or the furin-activated mature form?

MMP-11 is unusual among MMPs in that it is activated intracellularly by furin-type proprotein convertases before secretion, rather than extracellularly like most family members. This recombinant is expressed in HEK293 cells, which endogenously express furin, so the material is supplied predominantly as the mature, active enzyme rather than as an intact zymogen. This distinction is critical: you do not need to include an exogenous activator (e.g., APMA) in your activity assay setup, and any comparison with other MMP zymogens requiring activation should account for this difference in starting form.

What substrates does MMP-11 cleave and which is best for an in vitro activity assay?

MMP-11 has a relatively restricted substrate repertoire compared to other stromelysins. It cleaves α1-protease inhibitor (α1-PI/SERPIN A1) and IGFBP-1, among other extracellular matrix components, but has limited activity against the classic MMP substrates gelatin or casein. For a fluorogenic activity assay, Mca-KPLGL-Dpa-AR-NH2 or the generic MMP FRET substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 can be used, though turnover is moderate. A starting concentration of 5–25 nM recombinant MMP-11 and 10–50 µM fluorogenic peptide substrate in assay buffer is a reasonable range for dose-response or inhibitor IC50 experiments.

What assay buffer should I use for MMP-11 activity assays and does it differ from standard MMP buffers?

The storage buffer for this recombinant is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. For activity assays, dilute into a working buffer of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35, omitting glycerol at concentrations above 2–5% to avoid fluorescence artifacts in FRET-based readouts. Zinc is already coordinated in the active site; supplemental ZnSO4 is not typically required but 1–10 µM is sometimes added for long incubations. Keep CaCl2 present throughout — calcium is essential for MMP-11 structural stability and loss of activity in its absence is rapid.

What starting concentration of recombinant MMP-11 is recommended for an IC50 inhibitor screening assay?

For inhibitor screening, use 5–20 nM recombinant MMP-11 with a fluorogenic peptide substrate at or near its Km (typically 10–30 µM for Mca-KPLGL-Dpa-AR-NH2). At this enzyme concentration, the assay is sensitive enough to detect low-nanomolar inhibitors without substrate depletion artifacts over a 60-minute read window. Include EDTA (10 mM) and 1,10-phenanthroline (1 mM) as positive inhibitor controls, and DMSO vehicle at ≤1% (v/v) to avoid activity loss. Pre-incubate enzyme with inhibitor for 30 minutes at 37°C before adding substrate to allow equilibrium binding.

Can I use recombinant MMP-11 as a positive control for Western blot with the RP-MMP11 rabbit polyclonal antibody?

Yes — this is one of the primary use cases for pairing REC-MMP11 with RP-MMP11. The antibody and recombinant are produced in the same lab and are guaranteed compatible for this application. Load 20–50 ng of recombinant MMP-11 per lane on a 10–12% SDS-PAGE gel under reducing conditions. Expect a clean band at ~44–46 kDa (mature form) with RP-MMP11 at a working dilution of 1:500–1:2000, depending on your secondary antibody system. This positive control is especially useful when working with low-expressing cell lysates or FFPE tissue where endogenous MMP-11 signal may be weak.

How much recombinant MMP-11 should I load for a Western blot positive control lane?

20–50 ng per lane is the recommended starting range when pairing with the matched RP-MMP11 antibody (SKU: RP-MMP11). At 20 ng, the band is typically detectable with an HRP-conjugated secondary and standard ECL in 1–5 minutes of exposure. If you are titrating antibody concentration or comparing signal to cell lysate lanes, run a 3-point series at 10, 25, and 50 ng. Avoid loading more than 100 ng — excess recombinant can bleed into adjacent lysate lanes and complicate quantification if you are running samples and controls on the same gel.

How should I store and handle recombinant MMP-11 to preserve activity after receipt?

Upon receipt, spin briefly to collect liquid, then aliquot into single-use volumes before freezing at -20°C. The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — is formulated to maintain MMP-11 stability and metal coordination over multiple freeze-thaw cycles if avoided. Repeated freeze-thaw is the leading cause of activity loss for zinc-dependent proteinases; aliquots used intermittently should be stored at 4°C for no more than 7 days. Do not dilute to working concentrations in advance of the assay — prepare fresh dilutions in assay buffer on the day of use to minimize activity drift.