Anti-Archaemetzincin-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for AMZ1 on a Western blot?

The canonical human AMZ1 polypeptide is 498 amino acids with a predicted mass of approximately 55-57 kDa, and this is the band most users report at the 1/1000 working dilution. Migration can shift slightly depending on gel system and reducing conditions. Because AMZ1 carries an N-terminal propeptide, a lower-mass species corresponding to a processed/mature form may also appear in tissues with active proteolytic maturation. If you observe a band markedly above 60 kDa, consider post-translational modification or incomplete denaturation rather than off-target reactivity, and confirm by knockdown or peptide block.

Are there AMZ1 isoforms I should be aware of when interpreting bands?

UniProt Q400G9 lists the canonical 498 aa sequence as the primary isoform of human AMZ1. A shorter alternative transcript has been annotated, but characterisation at the protein level is limited and tissue distribution is not well established. In practice, most lysates show a dominant band near 55-57 kDa. If you see additional reproducible bands, distinguish isoform vs. propeptide processing vs. degradation by running fresh lysates with protease inhibitors and comparing to a recombinant AMZ1 control where available.

What is a good starting dilution for Western blot, and how should I optimise?

Start at 1/1000 in 5% non-fat milk or BSA/TBST, overnight at 4 °C, on 20-30 µg of total protein from a tissue or line with documented AMZ1 expression (e.g. testis, certain tumour lines). If signal is weak, titrate to 1/500 and extend exposure before increasing antibody. If background is high, move to BSA blocking and add 0.1-0.2% Tween-20 to washes. For IHC and IF the brand-wide validation applies, but optimal dilutions for those applications should be titrated against your fixation and antigen-retrieval conditions rather than extrapolated from the WB dilution.

What positive and negative controls are appropriate for AMZ1 detection?

Useful positive controls include testis lysate and tumour-derived lines reported to express AMZ1 in the original Díaz-Perales et al. (2005) characterisation and subsequent transcriptomic datasets; confirm expression in your specific model by RT-qPCR before committing to large blots. For negative controls, siRNA or shRNA knockdown of AMZ1 in the same line provides the strongest specificity evidence. A peptide competition with the immunogen, or comparison against an AMZ1-null tissue, can serve as orthogonal validation. Loading controls should be membrane-compatible housekeepers (GAPDH, β-actin, vinculin) appropriate for the 55-57 kDa range.

Will this antibody cross-react with mouse or rat AMZ1?

Reactivity is validated against human AMZ1. Mouse, rat, chimpanzee and other primate orthologues share substantial sequence identity across the metzincin catalytic core, so cross-reactivity is predicted but not experimentally validated in our hands. If you intend to use the antibody on rodent samples, run a parallel human positive control on the same blot and confirm the rodent band by knockdown or by comparison to recombinant mouse/rat AMZ1. Do not assume equivalent sensitivity across species, since the propeptide region is less conserved than the catalytic domain.

How do I distinguish AMZ1 from AMZ2 on a Western blot?

AMZ1 and AMZ2 are the two human archaemetzincin family members and share the conserved zinc-binding catalytic motif, but their overall sequence identity is moderate and their N- and C-terminal regions diverge. AMZ2 runs at a similar apparent mass (around 40-45 kDa for the canonical isoform), so size alone is not sufficient. The cleanest discrimination is genetic: AMZ1 knockdown should collapse the specific band without affecting AMZ2 transcript or protein. If you are working in a system that co-expresses both, run AMZ2-specific reagents in parallel rather than relying on migration.

What sample preparation works best for AMZ1?

Standard RIPA or 1% NP-40 lysis with complete protease inhibitors works well; AMZ1 is a soluble metalloprotease and does not require harsh membrane-solubilising detergents. Include EDTA in the lysis buffer if you want to suppress residual catalytic activity during extraction. Denature in Laemmli buffer with fresh reducing agent at 95 °C for 5 minutes; the antibody recognises a linear epitope and signal is reduced under non-reducing conditions. Avoid repeated freeze-thaw of lysates, which can generate sub-bands consistent with propeptide cleavage or non-specific proteolysis.

How should I store and handle the antibody to preserve activity?

The 100 µg vial ships in a standard polyclonal formulation (PBS with glycerol and sodium azide). Store at -20 °C for long-term use and aliquot on first thaw into low-binding tubes to avoid repeated freeze-thaw cycles, which are the most common cause of progressive signal loss on rabbit polyclonals. A working aliquot can be held at 4 °C for up to four weeks. Do not store at -80 °C in glycerol-containing buffer, since cycling through the glass transition can damage IgG. Spin briefly before pipetting to clear any aggregated material.