MMP-17 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant MMP-17 on SDS-PAGE and Western blot?

The full-length human MMP-17 protein is 603 amino acids with a predicted unmodified MW of ~67 kDa. The HEK293-expressed recombinant carries human-compatible N-linked glycosylation, so the observed band on reducing SDS-PAGE typically runs at 70–75 kDa. Under non-reducing conditions, you may see a slight upward shift due to disulfide-stabilized conformations. If you are running this alongside RP-MMP17 (our matched rabbit polyclonal), load the recombinant in a separate lane as a size reference — the glycosylated band will migrate distinctly above a deglycosylated or bacterially expressed comparator.

Which processing form of MMP-17 does this recombinant represent — zymogen or active enzyme?

REC-MMP17 is supplied as the active enzyme form, with the prodomain removed. Native MMP-17 is synthesized as an ~75 kDa zymogen and undergoes furin-mediated intracellular processing prior to GPI-anchor tethering at the cell surface. Because the recombinant lacks the GPI anchor (it is expressed as a soluble catalytic-domain-containing fragment), it is free in solution and enzymatically competent without additional activation steps. Researchers running zymogen-activation studies should note this distinction and not expect a pro-form band when using REC-MMP17 as a positive control.

What substrates does MMP-17 cleave and which fluorogenic substrate works best for activity assays?

MMP-17 has a relatively restricted substrate repertoire compared to other MT-MMPs. It cleaves fibronectin and gelatin, and has documented activity against the proteoglycan brevican. For in vitro fluorogenic activity assays, the broad-spectrum MMP substrate Mca-PLGL-Dpa-AR-NH2 (also sold as Peptide Substrate III) is the most widely used and gives reliable signal with REC-MMP17 at 1–10 nM enzyme concentration. Alternatively, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 is suitable if you need to distinguish MT-MMP activity. Use a 96-well black-walled plate and read fluorescence at Ex/Em 320/405 nm.

What buffer conditions should I use for MMP-17 activity assays and is CaCl2 required?

Yes, calcium is essential. MMP-17 is a zinc-dependent metalloproteinase that also requires Ca²⁺ for structural stability. REC-MMP17 is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂ — use this or a closely matched assay buffer. A standard activity assay buffer is 50 mM HEPES pH 7.0, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35. Avoid EDTA or EGTA entirely; even low-µM chelator contamination abolishes activity. ZnCl₂ supplementation (1–5 µM) is optional but can improve signal consistency across freeze-thaw cycles.

What starting concentration of recombinant MMP-17 should I use for an IC50 inhibitor assay?

For inhibitor IC50 determinations, a working enzyme concentration of 1–5 nM REC-MMP17 is appropriate when using Mca-PLGL-Dpa-AR-NH2 at 10 µM substrate. This places you well below substrate KM while maintaining a linear, measurable fluorescence rate over 30–60 minutes. Pre-incubate enzyme with inhibitor for 30 min at 37°C before adding substrate to allow equilibrium binding. If you are benchmarking against a broad MMP inhibitor such as GM6001 or NNGH, expect IC50 values in the low-nanomolar range, which serves as a useful activity verification step before testing novel compounds.

Can I use REC-MMP17 as a positive control for Western blot with the RP-MMP17 rabbit polyclonal antibody?

Yes — this is one of the primary use cases for REC-MMP17. The matched antibody RP-MMP17 (/anti-mmp-17-rabbit-polyclonal-antibody) was validated against this recombinant in-house, so band identity and signal intensity are well characterized. Load 10–50 ng REC-MMP17 per lane on a 10% polyacrylamide gel; the band at ~70–75 kDa will be cleanly detected with RP-MMP17 at a 1:1,000–1:2,000 dilution. Using the recombinant as a positive control is particularly valuable when screening low-expressing cell lysates where an endogenous band may be ambiguous.

How much recombinant MMP-17 should I load as a Western blot positive control for antibody validation experiments?

For antibody validation purposes using RP-MMP17, 20–50 ng per lane is the recommended loading range. At 20 ng, RP-MMP17 at 1:1,000 dilution produces a strong, clean band at ~70–75 kDa with minimal background under standard ECL detection. Titrating down to 5 ng is feasible with high-sensitivity substrates if you want to demonstrate antibody sensitivity. Include a no-primary-antibody lane as a negative control. Because REC-MMP17 and RP-MMP17 originate from the same production lot validation workflow, this pairing meets the antibody validation criteria outlined in the 2016 Nature Methods guidelines.

How should I handle, dilute, and store recombinant MMP-17 to maintain activity over time?

REC-MMP17 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 to protect enzymatic activity through a single freeze-thaw cycle; aliquot into single-use volumes before freezing to avoid repeated thawing. For working dilutions, use assay buffer (not water) to prevent activity loss from dilution-shock. Once thawed, keep on ice and use within 4 hours. Do not vortex; mix gently by pipetting. Shelf life at −20°C is 12 months from the date of manufacture when stored correctly.