MMP-13 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant MMP-13 on SDS-PAGE or Western blot?

The full-length MMP-13 proenzyme (UniProt P45452) has a predicted MW of ~54 kDa from its 471-aa sequence, but the HEK293-expressed recombinant typically runs at 55–60 kDa under reducing SDS-PAGE due to N-linked glycosylation. Upon activation and pro-domain removal (~10 kDa), the mature catalytic form migrates at approximately 42–48 kDa. If your experiment calls for the proenzyme form, verify your activation step is omitted. Under non-reducing conditions, expect a slight upward shift due to intact disulfide bonds. These values align with what you should see when using RP-MMP13 as your detection antibody.

What processing state is the recombinant MMP-13 supplied in — proenzyme or active enzyme?

REC-MMP13 is supplied as the active enzyme: the pro-domain has been removed during manufacturing, yielding the mature catalytic form. This means it is ready for direct use in activity assays without a separate activation step (e.g., APMA treatment). Researchers who specifically need the latent proenzyme form should note this distinction, as assay readouts measuring latent-to-active conversion would not be appropriate with this preparation. Purity is >95% by SDS-PAGE. If you need to confirm the activation state in your own hands, a 100 ng load resolved under reducing conditions and probed with RP-MMP13 should give a clean single band at ~42–48 kDa.

What substrates does MMP-13 cleave and which collagen type is cleaved most efficiently?

MMP-13 is a broad-spectrum collagenase with highest catalytic efficiency against soluble type II collagen, making it the primary driver of cartilage ECM degradation in osteoarthritis models. It also cleaves type I, III, IV, X, and XIV collagens, fibronectin, tenascin-C (TNC), and aggrecan (ACAN). Beyond structural matrix substrates, MMP-13 can process TGF-β1 and CCN2, positioning it at the boundary between matrix remodelling and growth-factor regulation. In fluorometric activity assays, a quenched fluorogenic peptide substrate such as Mca-PLGL-Dpa-AR-NH2 (type II collagen-mimetic cleavage site) is the standard choice for quantifying activity with this recombinant.

What buffer conditions should I use for a recombinant MMP-13 fluorogenic activity assay?

REC-MMP13 is supplied and stabilized in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol. For fluorogenic peptide cleavage assays, maintain Ca²⁺ at 1–5 mM in the assay buffer — calcium is required for catalytic domain stability and is not optional. A standard assay buffer is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 0.05% Brij-35. Avoid EDTA or EGTA; they chelate Zn²⁺ and Ca²⁺ and will abolish activity. ZnCl₂ supplementation at 1–10 µM is occasionally included but is not necessary if working from fresh aliquots.

What starting concentration of recombinant MMP-13 should I use for an in vitro activity assay and IC50 determination?

For fluorogenic peptide assays (e.g., Mca-PLGL-Dpa-AR-NH2 at 10 µM substrate), a starting enzyme concentration of 1–10 nM REC-MMP13 typically gives linear signal in the 30–60 min window on a standard plate reader. For IC50 determinations with MMP inhibitors, titrate enzyme to the lowest concentration yielding a robust S/N (≥5:1), commonly 2–5 nM, to avoid substrate depletion artifacts and to maintain conditions where [E] << [I] for accurate Ki estimation. Pre-incubate inhibitor with enzyme for 15–30 min at room temperature before adding substrate to allow equilibrium binding, particularly for slow-binding inhibitors.

Can I use REC-MMP13 as a positive control for Western blot with the RP-MMP13 antibody?

Yes — this is one of the primary validated use cases. REC-MMP13 and RP-MMP13 are produced and validated in the same lab, and compatibility for Western blot is confirmed. Load 50–200 ng of REC-MMP13 per lane under reducing conditions alongside your cell lysate or tissue samples. You should see a clean band at ~42–48 kDa (active form). RP-MMP13 is a rabbit polyclonal; use at the manufacturer-recommended dilution (typically 1:500–1:2000) with standard HRP-conjugated anti-rabbit secondary. This setup gives you an unambiguous positive control band to calibrate exposure and confirm antibody performance before interpreting endogenous signal.

How much recombinant MMP-13 should I load for a Western blot positive control lane?

50–200 ng per lane is the practical range. At 50 ng, RP-MMP13 at 1:1000 dilution will typically produce a strong, clean signal without overexposure; 100–200 ng is appropriate if you are pushing antibody dilution higher (1:2000) or using a less sensitive detection system. Because REC-MMP13 is the active, pro-domain-removed form, your positive control band will appear at ~42–48 kDa rather than at the ~55–60 kDa proenzyme position. If your biological samples are expected to contain the proenzyme, label the positive control lane clearly in your figure to avoid misinterpretation.

How should I store and handle recombinant MMP-13 to preserve activity — can I refreeze unused portions?

Store REC-MMP13 at -20°C in single-use aliquots. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 10% glycerol) stabilizes the enzyme through freeze-thaw, but repeated cycling will cause progressive activity loss — plan aliquot sizes around a single experimental session. Once thawed, keep on ice and use within 4–6 hours; do not return to -80°C. Avoid diluting into water alone; maintain Ca²⁺ and NaCl in any working dilution buffer. For long-term storage beyond 6 months, -80°C is preferable to -20°C to minimize ice-crystal damage over extended periods.