MMP-15 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant MMP-15 on SDS-PAGE or Western blot?

The full-length human MMP-15 protein (669 amino acids, UniProt P51511) has a predicted molecular weight of approximately 75 kDa. However, because this recombinant is produced in HEK293 mammalian cells, N-linked glycosylation adds mass, and the apparent MW on SDS-PAGE typically runs between 80–90 kDa under reducing conditions. If you are using this as a positive control with our matched rabbit polyclonal antibody RP-MMP15, expect a single prominent band in that 80–90 kDa range. Anomalous migration is common for MT-MMPs and does not indicate degradation.

What processing forms or isoforms of MMP-15 does this recombinant represent — propeptide, catalytic domain, or full-length?

REC-MMP15 is supplied as an active enzyme form in which the autoinhibitory propeptide has been removed, exposing the catalytic zinc-binding domain. It retains the hemopexin-like domain but lacks the transmembrane anchor, making it soluble in solution. This processed, soluble ectodomain closely mirrors the form generated by furin-mediated processing of native MT2-MMP at the cell surface. Researchers studying MMP-2 activation or ECM substrate cleavage in cell-free assays will find this the most relevant form, since the transmembrane anchor is not required for catalytic activity in vitro.

What substrates does MMP-15 cleave, and which fluorogenic substrate should I use for an activity assay?

MMP-15 is a broad-spectrum endopeptidase that cleaves ECM components including fibronectin, laminin, tenascin, and aggrecan. It also activates pro-MMP-2 by forming a complex with TIMP-2. For fluorogenic activity assays, the generic MMP substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (also called Knight substrate or FRET peptide I) is widely used and compatible with MMP-15. Excitation at 320 nm / emission at 405 nm. A typical starting concentration for REC-MMP15 is 10–50 nM enzyme in assay buffer, with substrate at 5–20 µM. Include 10 mM EDTA as a negative control to confirm zinc-dependent activity.

What assay buffer conditions are optimal for MMP-15 activity, and does the storage buffer interfere with fluorogenic assays?

REC-MMP15 is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. For fluorogenic or FRET-based activity assays, a working assay buffer of 50 mM HEPES pH 7.0–7.5, 150 mM NaCl, 10 mM CaCl2, 0.05% Brij-35 is standard and well-tolerated by MT-MMPs. Dilute REC-MMP15 at least 1:10 into assay buffer before use to bring glycerol below 1%, which can otherwise quench fluorescence and inhibit enzyme kinetics. Do not add EDTA or EGTA to working dilutions — both chelate the active-site zinc and fully abolish activity.

What starting concentration of recombinant MMP-15 should I use for inhibitor IC50 assays?

For IC50 determinations, use REC-MMP15 at 5–20 nM final concentration, with fluorogenic substrate (e.g., Mca-PLGL-Dpa-AR-NH2) at a concentration near the Km (typically 10 µM for MMP family substrates). Enzyme concentration should be kept at least 10-fold below the lowest inhibitor concentration tested to avoid tight-binding artifacts (the Morrison equation applies if you cannot maintain this ratio). Pre-incubate enzyme with inhibitor for 30–60 minutes at 37°C before initiating the reaction with substrate. Include a broad-spectrum MMP inhibitor such as GM6001 (1 µM) as a positive inhibition control in each plate.

Can I use REC-MMP15 as a positive control for Western blot with the RP-MMP15 antibody?

Yes — this is one of the primary validated uses of REC-MMP15. The matched rabbit polyclonal antibody RP-MMP15 (/anti-mmp-15-rabbit-polyclonal-antibody) was raised and validated against the same recombinant protein, ensuring direct compatibility. For Western blot positive controls, load 50–100 ng of REC-MMP15 per lane alongside your cell lysate samples. Under reducing SDS-PAGE conditions, RP-MMP15 detects a band at 80–90 kDa. This pairing is particularly useful for confirming antibody lot-to-lot consistency and for validating new secondary antibody systems before committing precious tissue lysates.

How much recombinant MMP-15 should I load for a Western blot positive control lane?

50–100 ng per lane is the recommended starting range for REC-MMP15 when used as a Western blot positive control alongside the matched RP-MMP15 antibody. At this loading, a clean band at approximately 80–90 kDa is reliably detected without signal saturation at standard antibody dilutions (RP-MMP15 is typically used at 1:500–1:2000 in 5% BSA/TBST). If your cell lysate samples are relatively MMP-15-sparse — as is common in non-tumor lines — a 100 ng spike lane serves as an unambiguous reference to confirm antibody performance and transfer efficiency.

How should I handle, aliquot, and store REC-MMP15 to maintain activity over time?

REC-MMP15 is shipped on dry ice and should be stored at -20°C immediately upon receipt. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) is formulated for long-term stability; purity is >95% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL. Prepare single-use aliquots before the first freeze to avoid repeated freeze-thaw cycles, which progressively reduce specific activity in metalloproteinases. Working dilutions should be prepared fresh in assay buffer on ice and used within 4 hours. Do not store diluted enzyme — glycerol is below the protective threshold once diluted.