MMP-24-H (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for MMP-24-H (Recombinant) on SDS-PAGE or Western blot?

The full-length human MMP-24 (UniProt Q9Y5R2) has a predicted MW of ~72 kDa, but this recombinant is expressed in HEK293 cells and carries native-like N-glycosylation, so the apparent MW on SDS-PAGE typically runs between 75–85 kDa under reducing conditions. The precise migration depends on your gel percentage and run conditions; a 10% polyacrylamide gel resolves it cleanly from background. Purity is confirmed at >95% by SDS-PAGE, so the dominant band is unambiguous. If you are running a Western blot positive control, pair with matched antibody RP-MMP24H for reliable detection.

What processing form is MMP-24 recombinant protein supplied in — zymogen or active enzyme?

REC-MMP24H is supplied as the active catalytic ectodomain, not the full-length zymogen. The prodomain has been removed during production, so no exogenous activation step (e.g., APMA or trypsin treatment) is required before use. This is the form relevant to studies of pericellular ECM remodelling and MMP-2 activation assays. Note that the transmembrane anchor is also absent, making this a soluble, ectodomain-only construct — appropriate for biochemical and cell-free activity experiments but not for reconstituting membrane-localized cleavage events.

What substrates can I use in a fluorescent activity assay with recombinant MMP-24-H?

MMP-24 cleaves a broad range of ECM substrates including fibronectin and chondroitin/dermatan sulfate proteoglycans, and activates pro-MMP-2. For fluorescent peptide assays, the generic MMP FRET substrate Mca-PLGL-Dpa-AR-NH2 (a.k.a. Knight substrate) is hydrolyzed by MMP-24 and provides a convenient continuous readout (Ex/Em ~325/393 nm). Substrate concentration of 10 µM in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10 µM ZnSO4) with 1–5 nM enzyme is a reasonable starting point. MMP-24 does not efficiently cleave collagen type I or laminin, so those substrates are not appropriate for positive-control activity assays.

What buffer should I use for MMP-24-H activity assays and is the storage buffer compatible?

REC-MMP24H is stored in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol. For activity assays, dilute the enzyme at least 1:10 into assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10 µM ZnSO4) to reduce glycerol to ≤1%, which can otherwise slightly attenuate kcat. Avoid EDTA or chelating agents in the assay buffer — even brief EDTA exposure will strip the catalytic zinc and permanently inactivate the enzyme. Keep pH between 7.0 and 7.8; MMP-24 activity drops sharply below pH 6.5.

What starting enzyme concentration should I use when measuring MMP-24 inhibitor IC50 values?

For inhibitor titration assays, start with 1–2 nM REC-MMP24H, keeping enzyme concentration well below the Ki of your inhibitor to satisfy steady-state kinetic assumptions and avoid tight-binding artifact. Use 10 µM Mca-PLGL-Dpa-AR-NH2 substrate (approximately Km for most MT-MMPs) and run the inhibitor series in triplicate. Include TIMP-2 as a positive inhibitor control (IC50 for MT-MMPs is typically in the low nanomolar range). Pre-incubate enzyme with inhibitor for 30 min at 37°C before substrate addition to allow equilibrium binding, especially for slow-on inhibitors.

Can I use MMP-24-H (Recombinant) as a Western blot positive control for the matched RP-MMP24H antibody?

Yes — this is one of the primary use cases for REC-MMP24H. Load 20–50 ng per lane alongside your cell lysate samples. The RP-MMP24H rabbit polyclonal antibody (/anti-mmp-24h-rabbit-polyclonal-antibody) was produced and validated in the same laboratory as this recombinant, and the two are guaranteed compatible for Western blot. At 50 ng loaded on a 10% gel, a clean band at ~75–85 kDa is expected after transfer to PVDF and primary antibody incubation at 1:500–1:1000 dilution. This approach also serves as a migration reference when interpreting endogenous MMP-24 bands in tissue or cell lysates.

How much recombinant MMP-24-H should I load as a positive control for antibody validation on Western blot?

20–50 ng per lane is sufficient for unambiguous detection with RP-MMP24H at a 1:500–1:1000 primary dilution. Loading above 100 ng risks saturating the signal and generating smearing artefacts, particularly given the >95% purity — essentially all loaded protein contributes to the target band. If you are co-running with crude cell lysate (which may contain endogenous MMP-24 at low levels), load the recombinant in an adjacent lane rather than mixing, to avoid interference with lysate band interpretation. HRP-conjugated anti-rabbit secondary at 1:5000 gives adequate sensitivity without excessive background.

How should I handle, dilute, and store MMP-24-H (Recombinant) to maintain activity over time?

REC-MMP24H is shipped as single-use aliquots and should be stored at -20°C immediately upon receipt. Avoid repeated freeze-thaw cycles — each cycle can cause measurable loss of metalloprotease activity due to zinc cofactor lability and protein aggregation. For short-term use (same week), aliquots may be held at 4°C for up to 5 days in storage buffer. When diluting for assay, use BSA-supplemented assay buffer (0.01–0.1% BSA) to prevent adsorptive losses at low protein concentrations. Do not dilute below 0.1 nM in the absence of carrier protein. Shelf life at -20°C is 12 months from the date of manufacture.