Anti-MMP-14 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for MMP-14 on a Western blot?

Full-length MMP-14 migrates at approximately 60–66 kDa, depending on glycosylation status. The protein contains an N-terminal signal peptide, propeptide, catalytic domain, hinge region, hemopexin-like domain, transmembrane domain, and short cytoplasmic tail. You may also observe bands around 45–50 kDa representing the active, processed form after propeptide removal, and lower molecular weight bands corresponding to shed ectodomains. Glycosylation heterogeneity can cause smearing or multiple bands. If working with cell lysates, include protease inhibitors to minimize autoproteolysis. Use a positive control such as HT-1080 fibrosarcoma or MDA-MB-231 breast cancer lysates, which express high endogenous MMP-14.

Does this antibody detect both the latent proenzyme and active MMP-14?

This polyclonal antibody is raised against residues spanning the extracellular portion of MMP-14, so it should recognize both the latent zymogen (with propeptide intact) and the activated, propeptide-cleaved form. Because rabbit polyclonals target multiple epitopes, detection does not depend on a single cleavage-sensitive region. Expect to see both the inactive pro-form and active enzyme on Western blots of cell or tissue lysates. If distinguishing activation status is critical, run parallel gels and probe with propeptide-specific or catalytic-domain-specific antibodies, or use functional assays such as MMP-2 activation zymography to confirm proteolytic activity in your samples.

Can I use this MMP-14 antibody for immunofluorescence on live cells?

MMP-14 is a type-I transmembrane protein with its catalytic and hemopexin domains exposed on the cell surface. If the epitope recognized by this polyclonal falls within the extracellular region, live-cell immunofluorescence is feasible without permeabilization. Incubate cells on ice with antibody diluted in serum-free medium to prevent internalization, then fix and apply fluorescent secondary. For intracellular or total MMP-14 staining, fix and permeabilize first. Triple Point Biologics antibodies are validated for immunofluorescence; start at 1:100–1:200 dilution and titrate. Use a known MMP-14-positive line such as HT-1080 as a control to optimize your protocol before experimental samples.

What is the recommended starting dilution for Western blot with this antibody?

The validated dilution for Western blot is 1:1000. This serves as a reliable starting point for most mammalian cell and tissue lysates. Load 20–50 µg total protein per lane and transfer to PVDF or nitrocellulose. Block with 5 percent non-fat milk or BSA in TBST for one hour at room temperature, then incubate overnight at 4°C with primary antibody. Because MMP-14 expression varies widely by cell type and activation state, you may need to adjust dilution and exposure time. High-expressing lines like HT-1080 or invasive cancer cells yield robust signals at 1:1000, whereas quiescent fibroblasts may require longer exposure or a more concentrated antibody solution.

Is this MMP-14 antibody cross-reactive with mouse and rat samples?

Human MMP-14 shares approximately 87 percent amino acid identity with mouse and 85 percent with rat orthologs, concentrated in the catalytic and hemopexin domains. Cross-reactivity with mouse and rat is listed as potential but not yet validated by Triple Point Biologics. If you plan to work with rodent samples, verify specificity by running a positive control of human lysate alongside mouse or rat lysate on the same blot. Check for bands at the expected molecular weight and confirm absence of signal in MMP-14 knockout tissue if available. Alternatively, consult the literature or perform a peptide-BLAST of the immunogen sequence against rodent MMP-14 to estimate epitope conservation before committing to large experiments.

What sample preparation steps minimize MMP-14 autoproteolysis in lysates?

MMP-14 is catalytically active at the cell surface and can undergo autoproteolysis or shed its ectodomain during lysis. Work quickly on ice and add a broad-spectrum protease inhibitor cocktail containing EDTA or EGTA to chelate zinc, the catalytic cofactor for MMPs. AEBSF or PMSF will block serine proteases but not MMPs; include a metalloproteinase inhibitor such as 10 mM EDTA, 10 µM GM6001, or 10 µM batimastat. Lyse cells in RIPA or NP-40 buffer supplemented with inhibitors, clarify by centrifugation, and snap-freeze aliquots immediately. Avoid repeated freeze-thaw cycles. For tissue samples, snap-freeze in liquid nitrogen immediately after harvest and homogenize under cryogenic conditions.

How should I store this MMP-14 antibody to maintain long-term stability?

Store the antibody at –20°C as supplied. The 100 µg vial is stable for at least 12 months under these conditions. For routine use, aliquot into single-use volumes (10–20 µL) immediately upon receipt to avoid repeated freeze-thaw cycles, which can aggregate IgG and reduce titer. Store working aliquots at –20°C or –80°C. If you use the antibody frequently, a 4°C stock in PBS with 0.02 percent sodium azide is acceptable for up to one month. Never store diluted antibody in blocking buffer for more than a few days, as carrier proteins can promote bacterial growth. Keep the original vial tightly sealed and desiccated to prevent condensation during temperature cycling.

Does MMP-14 expression vary between cytoplasmic and membrane fractions in Western blots?

MMP-14 is predominantly localized at the plasma membrane via its transmembrane anchor, but a fraction resides in intracellular vesicles, including endosomes and the trans-Golgi network, undergoing constitutive recycling. In a standard whole-cell lysate, you will detect both pools. If you fractionate, expect the majority of signal in membrane and light-membrane (vesicle) fractions, with minimal cytosolic signal unless ectodomain shedding has occurred. Use a membrane-enrichment kit or differential centrifugation, and validate fraction purity with markers such as Na-K-ATPase for plasma membrane and GAPDH or tubulin for cytosol. Membrane fractions may require stronger detergents such as 1 percent SDS or sonication for complete solubilization of MMP-14.