MMP-16 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant MMP-16 on SDS-PAGE or Western blot?

The full-length human MMP-16 (UniProt P51512) has a predicted MW of ~68 kDa from its 607-amino-acid sequence, but the recombinant form expressed in HEK293 cells typically migrates at approximately 70–75 kDa under reducing SDS-PAGE conditions, reflecting mammalian N-linked glycosylation. Under non-reducing conditions, migration may shift slightly depending on disulfide bond status. Purity is confirmed at >95% by SDS-PAGE. If you are running a Western blot positive control alongside our matched antibody RP-MMP16, expect a clean band in the 70–75 kDa range.

Does recombinant MMP-16 represent the full-length protein or a catalytic domain fragment?

This recombinant corresponds to the catalytic domain of MMP-16 rather than the full transmembrane-anchored 607-aa protein. The transmembrane anchor is omitted to enable soluble, active enzyme expression in HEK293 cells — a standard approach for MT-MMPs intended for in-solution biochemical assays. The catalytic domain retains the zinc-dependent endopeptidase activity characteristic of native MMP-16, including fibronectin and collagen type III cleavage, and supports pro-MMP-2 activation studies in cell-free systems. Researchers requiring the full-length membrane-localised context should use cell-based expression systems.

What substrates does recombinant MMP-16 cleave and which collagens should I avoid using in assays?

MMP-16 cleaves fibronectin and collagen type III with well-characterised efficiency and activates progelatinase A (pro-MMP-2) at the cell surface — both activities are retained by this recombinant catalytic domain. It does not cleave collagen types I, II, IV, or V under standard assay conditions, so these should not be used as positive substrate controls. For fluorescence-based activity assays, a broad-spectrum MMP FRET substrate such as Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 is appropriate for confirming lot activity before advancing to ECM substrate panels.

What assay buffer should I use for MMP-16 activity assays and does the storage buffer interfere?

MMP-16 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, and 5 mM CaCl₂. For activity assays, dilute into a standard MMP assay buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, and 0.05% Brij-35 — the detergent reduces non-specific surface adsorption without inhibiting the zinc-active site. Keep CaCl₂ at ≥1 mM throughout; calcium depletion rapidly destabilises the catalytic domain. At final working dilutions (≥10-fold), residual glycerol from the storage buffer does not measurably affect fluorometric readouts.

What starting concentration of recombinant MMP-16 should I use for a fluorometric FRET activity assay?

A starting concentration of 50–200 nM recombinant MMP-16 in a 100 µL assay volume is a practical working range with a 10 µM FRET substrate (e.g., Mca-PLGL-Dpa-AR-NH₂). Run an enzyme titration at 25, 50, 100, and 200 nM to identify the linear range for your substrate and plate reader. Because lot-to-lot specific activity can vary within the >95% purity specification, confirming activity on receipt with a small pilot assay before committing samples to inhibitor IC₅₀ experiments is advisable. The 5 mM CaCl₂ in the storage buffer is compatible with direct dilution into most MMP assay buffers.

How do I account for MMP-16 glycosylation when running inhibitor binding or IC50 experiments?

Because this recombinant is expressed in HEK293 cells, the catalytic domain carries mammalian N-linked glycosylation, which supports the native active-site geometry relevant to inhibitor binding studies — a meaningful advantage over E. coli-derived or deglycosylated preparations where active-site conformation can be compromised. When calculating IC₅₀ values, confirm that the inhibitor concentration range brackets at least two orders of magnitude around the expected Ki, and hold enzyme concentration well below the Ki to avoid tight-binding artefacts. Substrate and enzyme concentrations should be validated in the linear range before advancing to inhibitor titrations.

Can I use recombinant MMP-16 as a Western blot positive control with the RP-MMP16 antibody?

Yes — REC-MMP16 is the intended positive control for RP-MMP16. Load 10–50 ng of recombinant protein per lane under reducing conditions; 20 ng is a reliable starting point for most chemiluminescence detection systems. Expect a band at ~70–75 kDa. RP-MMP16 is a rabbit polyclonal validated for Western blot at Triple Point Biologics, and both products were developed from the same research program, ensuring epitope–protein compatibility. This pairing is particularly useful for validating antibody performance against cell lysates where endogenous MMP-16 expression is low or variable.

How much recombinant MMP-16 should I load alongside RP-MMP16 antibody for antibody validation on Western blot?

For antibody validation experiments using RP-MMP16, loading a titration of 5, 20, and 50 ng of REC-MMP16 per lane is the most informative approach: it demonstrates both sensitivity (lowest detectable amount) and linearity of the antibody signal. Run alongside your cell lysate of interest so the recombinant band at ~70–75 kDa serves as an unambiguous size marker for the endogenous protein. This three-point titration typically requires less than 200 ng total recombinant protein, well within a single aliquot, and provides publication-quality documentation of antibody specificity.

How should I store recombinant MMP-16 and what is the recommended shelf life after aliquot thaw?

Store REC-MMP16 at -20°C in the supplied single-use aliquots. The 10% glycerol in the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂) acts as a cryoprotectant and ensures protein stability through multiple freeze-thaw cycles is not necessary — aliquots are sized to avoid this entirely. Once thawed, keep on ice and use within 24 hours; do not refreeze. Residual activity after extended incubation at 4°C degrades measurably beyond 48 hours. Under recommended conditions, shelf life from manufacture is 12 months.