MMP-19 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant MMP-19 on SDS-PAGE or Western blot?

The full-length human MMP-19 precursor runs at approximately 57–60 kDa on reducing SDS-PAGE, consistent with the 508-amino-acid sequence (UniProt Q99542) plus HEK293-derived N-linked glycosylation. The processed, catalytically active form — lacking the signal peptide and propeptide — typically migrates near 45–48 kDa. Because this recombinant is expressed in HEK293 cells, glycoforms can produce a slightly diffuse band. Running under reducing conditions and including REC-MMP19 alongside your lysate samples gives a clean reference ladder point when probing with RP-MMP19.

What is the processing state of REC-MMP19 — is it the zymogen or the active catalytic form?

REC-MMP19 is supplied as the active enzyme, with the autoinhibitory propeptide removed or processed during HEK293 expression and purification. It retains both the catalytic zinc-binding domain and the C-terminal hemopexin-like domain, preserving the full-length active architecture relevant to substrate engagement and inhibitor binding. This distinguishes it from truncated catalytic-domain-only constructs. If your assay requires the zymogen form for activation experiments, note that REC-MMP19 is not suitable for that specific workflow; it is intended for direct activity and inhibitor profiling.

Which substrates does MMP-19 cleave, and which are best for a fluorogenic activity assay?

MMP-19 has documented activity against aggrecan, COMP, collagen type IV, laminin, nidogen, tenascin-C isoforms, fibronectin, and type I gelatin. For fluorogenic activity assays in vitro, the generic MMP fluorogenic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ (or equivalent FRET-quenched peptides based on the conserved MMP cleavage consensus) works reliably as a first-pass screen. For physiologically relevant substrate assays, type I gelatin and collagen IV degradation assays are well-established entry points. Confirm substrate concentration is not enzyme-rate-limiting by running a brief Km estimation before committing to inhibitor IC₅₀ work.

What buffer conditions should I use for MMP-19 activity assays, and does calcium matter?

REC-MMP19 is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂. For activity assays, the storage buffer is compatible as-is, or you can dilute into a standard MMP assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35). Calcium is essential — MMP family members require Ca²⁺ for structural stability of the catalytic domain, and omitting it rapidly reduces activity. Keep assay pH between 7.0–7.5; activity drops measurably below pH 6.5 or above 8.0. Avoid EDTA or EGTA in assay buffers, as both will chelate the active-site zinc.

What starting concentration of recombinant MMP-19 should I use for a fluorogenic inhibitor IC50 assay?

A practical starting point is 1–5 nM REC-MMP19 in the assay well, which typically yields a robust signal-to-background ratio with standard Mca-quenched MMP substrates at 10–20 µM. Confirm that less than 10% of substrate is consumed at endpoint (the "10% rule") to maintain steady-state kinetics. If you are working with a tight-binding inhibitor (Ki < 10 nM), keep enzyme concentration at or below your expected Ki to avoid stoichiometric binding artifacts that compress the apparent IC₅₀. Titrate enzyme concentration empirically on your fluorescence reader before committing to an inhibitor dilution series.

Can I use REC-MMP19 as a positive control for Western blot with the RP-MMP19 antibody?

Yes — this is a primary use case. REC-MMP19 and RP-MMP19 are sourced from the same production pipeline at Triple Point Biologics, with antibody immunogen design matched to the recombinant antigen, making this the most direct positive control pairing available. Load 20–50 ng of REC-MMP19 per lane on a standard 10% SDS-PAGE gel under reducing conditions. Expect a band at approximately 45–60 kDa (active form to glycosylated precursor range). This confirms antibody performance and establishes the expected band position before interpreting endogenous MMP-19 signals in cell lysates or tissue extracts.

How much recombinant MMP-19 should I load for a Western blot positive control lane?

Load 20–50 ng per lane as a starting point when using RP-MMP19 as the detection antibody. At 20 ng, a well-optimized ECL detection should yield a clear, non-saturated band in the 45–60 kDa region. If your lysate lanes require a longer exposure, scale the recombinant load down to 5–10 ng to keep the positive control in the linear detection range and avoid blooming that obscures nearby markers. Running a two- or three-point dilution series of REC-MMP19 (10, 25, 50 ng) alongside lysates in initial experiments helps bracket the quantitative range for semi-quantitative comparisons.

How should I store and handle REC-MMP19 to preserve enzymatic activity, and what is the shelf life?

REC-MMP19 is shipped 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 — each cycle can reduce specific activity by 15–30% in metalloproteinase preparations. When thawing, place the aliquot on ice and use within the same working day. Do not dilute the working stock more than 24 hours in advance; prepare fresh dilutions in assay buffer containing at least 5 mM CaCl₂ and 0.05% BSA (carrier protein) to minimize surface adsorption at low concentrations. Shelf life is 12 months from receipt at −20°C.