MMP-24-M (Recombinant)

Frequently Asked Questions

What molecular weight band does recombinant MMP-24-M run at on SDS-PAGE or Western blot?

The full-length MMP-24 sequence is 645 amino acids (UniProt Q9Y5R2), predicting an unmodified MW of ~71 kDa. However, because REC-MMP24M is expressed in HEK293 cells, N-linked glycosylation adds mass, and the apparent MW on reducing SDS-PAGE typically migrates closer to 75–80 kDa. If you are using our matched antibody RP-MMP24M for Western blot, expect your positive control band in that 75–80 kDa range. Under non-reducing conditions, intermolecular disulfide artifacts can shift the apparent band further — use fresh β-mercaptoethanol or DTT to confirm.

What processing or activation state is recombinant MMP-24-M supplied in — zymogen or active form?

REC-MMP24M is supplied as the active enzyme, not the latent zymogen. The prodomain has been removed during production, so the catalytic domain is accessible without additional activation steps such as APMA treatment. As a membrane-type MMP, MMP-24 is naturally processed at the cell surface; this recombinant reflects that processed, active state. If your assay requires defined latency controls alongside active enzyme, we recommend preparing a separate APMA-treated sample from a zymogen source rather than using this material for that purpose.

What substrates does MMP-24 cleave and which fluorogenic peptide substrate works best for activity assays?

MMP-24 cleaves components of the extracellular matrix including aggrecan, fibronectin, and gelatin, and has been shown to shed cell-surface proteoglycans. For fluorogenic activity assays, the generic MMP substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ (also sold as MMP FRET Substrate I) provides a reliable signal with MT-MMPs at excitation/emission ~320/405 nm. As a starting point, use 10 µM substrate with 0.5–2 nM REC-MMP24M in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 0.05% Brij-35) at 37°C. Validate linearity across your concentration range before comparing inhibitor IC₅₀ values.

What buffer conditions are optimal for MMP-24-M enzymatic activity assays and does calcium matter?

The storage buffer for REC-MMP24M is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂. Calcium is essential — MMP-24, like all MMPs, requires Ca²⁺ for structural stability of the catalytic domain, and chelation by EDTA or EGTA will abolish activity. For activity assays, dilute into assay buffer matching the storage buffer composition but omitting glycerol and adding 0.05% Brij-35 to reduce surface adsorption. Maintain pH between 7.0 and 7.5; activity drops measurably below pH 6.8. Zinc supplementation (1–10 µM ZnCl₂) can improve signal consistency in extended incubations.

What starting concentration of recombinant MMP-24-M should I use for an inhibitor IC50 assay?

For IC₅₀ determination, enzyme concentration should be well below the substrate Km to satisfy tight-binding assumptions and avoid substrate depletion artifacts. A working range of 0.5–2 nM REC-MMP24M with 10 µM fluorogenic substrate is appropriate as an initial titration point. Confirm that RFU increase is linear over your assay window (typically 30–60 min at 37°C) before adding inhibitor. At higher enzyme loads (>5 nM), substrate depletion within the assay window can compress apparent IC₅₀ values. Include a broad-spectrum MMP inhibitor such as GM6001 (1 µM) as a positive inhibition control in every plate.

Can I use REC-MMP24M as a positive control for Western blot with the matched antibody RP-MMP24M?

Yes — that is the primary design intent of the pairing. RP-MMP24M (see /anti-mmp-24m-rabbit-polyclonal-antibody) is a rabbit polyclonal antibody validated for Western blot against this recombinant, so the two are guaranteed compatible. Load 20–50 ng of REC-MMP24M per lane on a standard 10% SDS-PAGE gel under reducing conditions. This produces a clean band at ~75–80 kDa that serves both as a size marker for endogenous MMP-24 in your cell lysates and as a sensitivity reference for antibody dilution optimization. Run alongside your lysate to confirm band co-migration before committing to a full experimental set.

How much recombinant MMP-24-M should I load for Western blot positive control and what antibody dilution is recommended?

Load 20–50 ng of REC-MMP24M per lane for a strong, unambiguous positive control band. For the matched antibody RP-MMP24M, start at a 1:1000 dilution in 5% non-fat milk/TBST for primary incubation (overnight, 4°C); titrate to 1:2000 if background is high in your specific membrane system. The recombinant runs at ~75–80 kDa under reducing conditions. If you are loading total cell lysate alongside, 10–20 ng of recombinant is usually sufficient to distinguish the spiked band from endogenous signal without overwhelming the lane. Confirm with Ponceau staining to verify equal transfer before immunodetection.

How should I handle, dilute, and store REC-MMP24M to preserve enzymatic activity over time?

REC-MMP24M is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂ and is stored at −20°C in single-use aliquots. Repeated freeze-thaw cycles measurably reduce specific activity — do not refreeze working aliquots. When diluting to low concentrations (<10 nM) for activity assays, use carrier protein (0.1% BSA, protease-free) in your dilution buffer to prevent adsorption losses to plastic surfaces. On ice, the enzyme is stable for 4–6 hours; avoid prolonged incubation at 37°C without substrate present. Purity is >90% by SDS-PAGE and endotoxin is <0.1 EU/µg, so no additional clean-up steps are needed before functional assays.