Archaemetzincin-2 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant Archaemetzincin-2 on SDS-PAGE or Western blot?

The full-length AMZ2 sequence encodes 360 amino acids, giving a calculated molecular weight of approximately 40.5 kDa. Because this recombinant is produced in HEK293 mammalian cells, potential N-glycosylation can shift the apparent band on SDS-PAGE to roughly 42–46 kDa depending on gel system and running conditions. Under reducing conditions you should observe a single predominant band above the 40 kDa marker. If you see a doublet, this likely reflects differential glycosylation rather than proteolytic processing or a contaminating species.

Is recombinant AMZ2 expressed as full-length or a processed isoform, and does it require activation?

This preparation covers the complete 360-residue sequence of human AMZ2 (UniProt Q86W34) and is supplied as the active enzyme — no propeptide removal or exogenous activation step is required before use. AMZ2 lacks the canonical pro-domain found in many metzincins, consistent with how Díaz-Perales et al. (J Biol Chem, 2005) originally characterized the archaemetzincin family. The zinc-binding HEXXH motif is intact, and HEK293 expression ensures proper metal coordination. If your assay format demands a defined activation state, confirm zinc saturation by including 1–5 µM ZnCl₂ in your pre-incubation step.

What substrates can I use to measure Archaemetzincin-2 protease activity in vitro?

AMZ2's precise endogenous substrate repertoire remains under active investigation, but as a zinc metalloprotease it cleaves synthetic fluorogenic peptide substrates containing the Mca/Dnp FRET pair widely used for metzincin family members — for example, Mca-PLGL-Dpa-AR-NH₂. Start at 10–50 µM substrate with 50–200 nM AMZ2 in the supplied storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 10% glycerol). Monitor fluorescence at Ex/Em 320/405 nm. Include a metalloprotease inhibitor such as 1,10-phenanthroline (5 mM) as a negative control to confirm zinc-dependent activity.

What buffer conditions are optimal for Archaemetzincin-2 activity assays, and can I dilute into my own buffer?

The supplied storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 10% glycerol — is compatible with direct use in activity assays. For dilution into an assay buffer, maintain CaCl₂ at 1–5 mM and keep pH between 7.0 and 7.8; activity drops noticeably below pH 6.5. Avoid EDTA or EGTA, which will chelate the catalytic zinc and abolish activity. Keep glycerol above 5% if diluting substantially, to prevent surface adsorption. BSA (0.01–0.1%) can be added as a carrier when working below 50 nM enzyme.

What starting concentration of recombinant AMZ2 should I use for an initial IC50 inhibitor screen?

For a preliminary IC50 experiment, 50–100 nM AMZ2 is a practical starting point with a fluorogenic FRET substrate at 20–30 µM (well below estimated Km to stay in the linear range). This concentration gives a robust signal-to-background ratio while keeping enzyme consumption manageable across a full inhibitor dilution series. Confirm that substrate conversion remains below 10–15% at your chosen time-point to maintain steady-state kinetics. If you are using a tight-binding inhibitor, titrate enzyme down to 10–20 nM and apply Morrison quadratic fitting rather than standard Cheng-Prusoff correction.

Can I use recombinant Archaemetzincin-2 as a positive control for Western blot with the matched TPB antibody RP-Archaemetzincin2?

Yes — this is the recommended use. REC-Archaemetzincin2 and the matched rabbit polyclonal RP-Archaemetzincin2 (/anti-archaemetzincin-2-rabbit-polyclonal-antibody) are validated together in-house for Western blot. Load 20–50 ng of recombinant protein per lane under reducing conditions; the antibody detects the ~42–46 kDa glycosylated band reliably at that range. This pairing is also useful for antibody lot qualification: a consistent signal at the expected MW with no additional major bands confirms antibody specificity before you commit it to endogenous tissue lysates.

How much recombinant Archaemetzincin-2 should I load as a positive control for Western blot, and what antibody dilution works?

Load 20–50 ng per lane for a clean, quantifiable band without signal saturation. Using RP-Archaemetzincin2 at 1:500–1:2000 dilution in 5% non-fat milk or BSA/TBST, expect detection within a standard 1-hour secondary incubation with an HRP-conjugated anti-rabbit antibody. If your cell lysate lanes require a longer exposure, reduce the recombinant positive control to 10 ng to keep it in a comparable dynamic range. The matched antibody-recombinant pair has been validated by the same TPB laboratory that has produced metalloprotease reagents since 1994, so lot-to-lot consistency is tightly controlled.

How should I handle, aliquot, and store recombinant AMZ2 to maintain activity over time?

REC-Archaemetzincin2 is supplied in single-use aliquots in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 10% glycerol. Store at -20°C and avoid repeated freeze-thaw cycles, which degrade metalloprotease activity through zinc loss and aggregation. On the day of use, thaw on ice and dilute immediately into assay buffer; do not leave the protein at room temperature for extended periods. If you need to prepare working stocks below 50 nM, add 0.01% BSA to the dilution buffer to minimize adsorption losses. Shelf life is 12 months from receipt when stored correctly.