Anti-MMP-16 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for MMP-16 on Western blot?

Full-length MMP-16 (MT3-MMP) runs at approximately 64 kDa on reducing SDS-PAGE, corresponding to the 607-amino acid membrane-anchored form. You may also detect a prominent band near 52-55 kDa representing the shorter isoform, which retains fibronectin and collagen III cleavage activity. In some cell lysates, particularly from melanoma or vascular tissues with active remodeling, you may observe additional bands corresponding to processed or shed forms. The banding pattern depends on cell type, activation state, and whether you are analyzing whole-cell lysates versus membrane-enriched fractions. Always include a known MMP-16-expressing cell line as a positive control.

What starting dilution should I use for MMP-16 Western blot detection?

We recommend starting at 1:1000 dilution in blocking buffer containing 5 percent non-fat dry milk or BSA in TBST for overnight incubation at 4°C. This polyclonal antibody performs well within the 1:500 to 1:2000 range, but optimal dilution depends on MMP-16 expression level in your sample. Membrane-type MMPs like MMP-16 are often enriched in membrane fractions, so cytosolic preparations may require higher antibody concentration or longer exposure. Titrate against your specific lysate type and include a lysate from HT-1080 fibrosarcoma or melanoma cells as a high-expression control to bracket signal.

Does this MMP-16 antibody distinguish between the full-length and shorter isoforms?

This polyclonal antibody was raised against a region spanning residues within the catalytic and hinge domains, so it recognizes both the full-length 607-amino acid membrane-anchored form and the shorter isoform that retains fibronectin and collagen III cleavage activity. You will typically detect both on Western blot as distinct bands near 64 kDa and 52-55 kDa. If your experiment requires isoform-specific detection, you will need to confirm the epitope map or select an antibody targeting the unique C-terminal region present only in the full-length protein. For most extracellular matrix remodeling studies, detecting both isoforms is informative.

Is this antibody validated for mouse and rat MMP-16 detection?

Human MMP-16 has been validated experimentally with this antibody. Mouse and rat cross-reactivity are predicted based on sequence homology in the immunogen region but have not been formally validated in our hands. Human and mouse MMP-16 share approximately 85 percent sequence identity overall, with higher conservation in the catalytic domain. If you are working with rodent samples, plan to titrate the antibody and include a positive control such as transfected lysate or tissue known to express MMP-16, like aorta or tumor xenografts undergoing active remodeling. Empirical testing is the only way to confirm signal specificity in non-validated species.

What sample preparation is best for detecting membrane-bound MMP-16?

Because MMP-16 is a membrane-type MMP anchored to the plasma membrane via a transmembrane domain, enrichment improves sensitivity. Use RIPA or 1 percent Triton X-100 lysis buffer supplemented with protease inhibitors; avoid mild lysis conditions that leave membranes intact. For maximum yield, consider membrane fractionation kits or ultracentrifugation protocols that pellet membrane fractions. Load 20-40 micrograms of total protein per lane or 5-10 micrograms of membrane-enriched lysate. Avoid boiling samples excessively, as this can aggregate membrane proteins; heating at 70°C for 10 minutes in reducing sample buffer is sufficient for most applications.

Can I use this antibody for immunohistochemistry on tissue sections?

Yes, This antibody is validated for Western blot; IHC application data is in progress. For paraffin-embedded sections, antigen retrieval is typically required; citrate buffer (pH 6.0) heat-induced epitope retrieval works well for most MT-MMPs. Use the antibody at dilutions between 1:100 and 1:400 depending on tissue MMP-16 expression and detection system sensitivity. MMP-16 localizes to the cell surface and invading front in tumors, and to endothelial and smooth muscle layers in vascular tissue. Include appropriate negative controls such as primary antibody omission and isotype control. For frozen sections, fixation with 4 percent paraformaldehyde or acetone generally preserves epitope accessibility without retrieval.

How should I store this MMP-16 antibody to maintain activity?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase background. The antibody is supplied at 100 micrograms in a stabilized buffer; upon receipt, aliquot into working volumes suitable for 4-6 experiments to minimize freeze-thaw. For short-term use within 1-2 weeks, diluted antibody can be stored at 4°C in the presence of 0.02 percent sodium azide, though we recommend preparing fresh dilutions for each experiment when possible. Do not store diluted antibody long-term without preservative. Properly stored aliquots remain stable for at least 12 months at -20°C.

What are suitable positive and negative controls for MMP-16 experiments?

For positive controls, use lysates from HT-1080 fibrosarcoma cells, melanoma cell lines such as A375, or primary human vascular smooth muscle cells stimulated with TGF-beta or PDGF, all of which express MMP-16 during extracellular matrix remodeling. Membrane fractions from these cells yield the strongest signal. For negative controls, use cell lines with minimal or absent MMP-16 expression, or pre-incubate the antibody with blocking peptide if available. Knockdown or knockout lysates provide the best specificity control. In IHC, normal tissues with low MMP-16 such as liver or kidney cortex serve as useful negative anatomical controls, while tumor invasion fronts or atherosclerotic plaques are positive.