Anti-MMP-15 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for MMP-15 on a Western blot?

MMP-15 (MT2-MMP) runs at approximately 65-72 kDa on Western blot, corresponding to the full-length membrane-anchored form. The 669-amino acid protein includes a signal peptide, propeptide, catalytic domain, hinge region, hemopexin-like domain, transmembrane domain, and short cytoplasmic tail. You may observe the zymogen (pro-MMP-15) around 68 kDa and the activated form around 60 kDa following propeptide cleavage. Some samples show additional bands representing glycosylated variants or proteolytic fragments. Cell lysates should be prepared under reducing conditions to maintain the full-length protein for detection.

Does MMP-15 require membrane protein extraction for Western blot detection?

Yes, because MMP-15 is a membrane-type MMP with a C-terminal transmembrane domain, standard RIPA buffer may underextract it. For optimal recovery, use lysis buffers containing 1% SDS or include additional detergent (1-2% Triton X-100 or NP-40). Membrane protein extraction kits improve yield significantly. If using whole-cell lysates, increase protein loading to 30-50 µg per lane rather than the typical 20 µg. Alternatively, enrich membrane fractions by ultracentrifugation. The recommended 1:1000 dilution works well once membrane proteins are adequately solubilized. Include a positive control such as HT-1080 fibrosarcoma or HeLa lysate.

Is this MMP-15 antibody specific or does it cross-react with other MMPs?

This rabbit polyclonal was raised against MMP-15-specific epitopes and has been validated for human MMP-15. MMPs share considerable domain homology, particularly within the catalytic and hemopexin regions, so always confirm specificity in your system. Run a Western blot with lysates from cells known to express different MMP family members, or use siRNA knockdown of MMP-15 to verify the band disappears. MMP-15 is distinct as a membrane-type MMP, which helps differentiate it from secreted forms. Cross-reactivity with MMP-14 (MT1-MMP) or MMP-16 (MT3-MMP) is possible given structural similarity; validate with appropriate positive and negative controls.

What starting dilution should I use for IHC with this MMP-15 antibody?

For immunohistochemistry, start with a 1:100 to 1:200 dilution and titrate based on signal-to-noise in your tissue type. Antigen retrieval is typically required; citrate buffer (pH 6.0) at high temperature for 20 minutes works well for most formalin-fixed paraffin-embedded tissues. Because MMP-15 is membrane-localized, expect staining at cell surfaces and pericellular regions rather than diffuse cytoplasmic signal. Tissues with known MMP-15 expression—such as invasive carcinoma margins, endothelial cells during angiogenesis, or remodeling connective tissue—serve as useful positive controls. Optimize blocking conditions to reduce background in high-protease environments.

Does this antibody detect mouse or rat MMP-15 for in vivo studies?

The antibody is validated for human MMP-15; mouse and rat reactivity are predicted but not experimentally confirmed. Human and mouse MMP-15 share approximately 80-85% amino acid identity, with the highest conservation in the catalytic domain. If you plan to use mouse or rat samples, validate reactivity first with a positive control lysate from cells overexpressing rodent MMP-15 or tissue known to express it endogenously, such as placenta or invasive tumor models. Run Western blots alongside human samples to compare band patterns. Cross-reactivity is likely but should be empirically confirmed for publication-quality data.

What are appropriate positive controls for MMP-15 Western blot experiments?

HT-1080 fibrosarcoma cells constitutively express MMP-15 and serve as a reliable positive control. HeLa, MCF-7, and certain melanoma lines also express detectable levels. For negative controls, use cells with minimal MMP-15 expression or perform siRNA-mediated knockdown to confirm band specificity. Because MMP-15 expression is often induced during cell migration or invasion, consider stimulating cells with TGF-β, TNF-α, or culturing in three-dimensional matrices to upregulate expression. Recombinant MMP-15 protein is an additional control option, though the catalytic domain alone may run at a different molecular weight than full-length membrane-anchored native protein.

How should I store the MMP-15 antibody and what is its shelf life?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100 µg pack size allows convenient aliquoting: dilute a portion in storage buffer containing 50% glycerol and 0.02% sodium azide for a working stock that remains liquid at -20°C, or make 10 µL aliquots of the concentrated stock. Once thawed, an aliquot can be stored at 4°C for up to one month if sodium azide is present. Do not store diluted antibody in working solutions for more than a week. Shelf life is typically two years from manufacture when stored properly.

Can I use this antibody to detect secreted or soluble MMP-15 forms?

MMP-15 is a membrane-type MMP and physiologically remains cell-surface-tethered via its transmembrane domain, so it is not secreted like MMP-2 or MMP-9. However, proteolytic shedding by sheddases or alternative splicing can release a soluble ectodomain lacking the transmembrane anchor. If you are studying conditioned media or serum, concentrate samples by precipitation or ultrafiltration and run Western blots, but expect lower abundance than cell-associated forms. The antibody epitope location determines whether it detects shed fragments; if raised against the extracellular domains, it should recognize soluble forms. Always include cell lysate positive controls for comparison.