Meprin A (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant Meprin A alpha on SDS-PAGE or Western blot?

The MEP1A precursor is 746 amino acids (~85 kDa predicted), but the HEK293-expressed recombinant runs at approximately 90–100 kDa under denaturing conditions due to N-glycosylation. Under non-reducing conditions, disulfide-linked dimers or higher-order complexes may appear above 150 kDa. If you are using this as a positive control alongside RP-MeprinA, load the reducing lane first to confirm the ~95 kDa glycoprotein band before interpreting non-reduced blots.

What processing form of Meprin A does this recombinant represent — zymogen, prodomain-cleaved, or mature enzyme?

This recombinant is the active, prodomain-processed form of human Meprin A alpha (Q16819). The inhibitory prodomain is removed during HEK293 expression and downstream processing, yielding the mature catalytic enzyme. It is not a zymogen. Because the propeptide is absent, no exogenous activation step (e.g., trypsin treatment) is required prior to activity assays. If your experiment requires a latent zymogen control, this product is not suitable for that purpose.

What substrates does recombinant Meprin A alpha cleave, and are there validated fluorogenic peptide substrates for activity assays?

Meprin A alpha (EC 3.4.24.18) is a zinc-dependent astacin-family endopeptidase with a preference for acidic residues at P1'. Well-validated fluorogenic substrates include the quenched peptide Mca-RPPGFSAFK(Dnp)-OH and OCT (octa-peptide) variants containing acidic cleavage sites. Pro-collagen, IL-18, and fibronectin fragments are documented protein substrates. For initial activity confirmation, 10 µM Mca-RPPGFSAFK(Dnp)-OH in the standard assay buffer at 37 °C is a practical starting point. Monitor fluorescence at Ex/Em 320/405 nm.

What assay buffer should I use for Meprin A (Recombinant) activity assays, and does storage buffer interfere?

Meprin A alpha is active in the pH 6.5–7.5 range; 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 (matching the storage buffer) works well and avoids a dilution-induced buffer switch. Calcium is required for catalytic stability — do not use EDTA or EGTA-containing buffers, as these chelate the active-site zinc and will abolish activity. If your assay buffer is different, dilute the recombinant at least 1:10 into assay buffer immediately before use to minimize glycerol carry-over (final glycerol <1%).

What starting concentration of recombinant Meprin A should I use in a metalloproteinase activity assay?

For fluorogenic peptide cleavage assays (e.g., Mca-RPPGFSAFK(Dnp)-OH at 10 µM), 1–5 nM recombinant Meprin A alpha is a practical starting range; most labs find a linear response between 0.5 and 10 nM enzyme at 37 °C over 30–60 minutes. For substrate Km determination, titrate enzyme at 2 nM with substrate from 1–100 µM. For cell-based or tissue homogenate assays, 10–50 nM is typical to achieve a signal above background within 60 minutes.

How do I factor in IC50 measurements for Meprin A inhibitors using this recombinant?

Use a fixed enzyme concentration of 2–5 nM and a substrate concentration at or below the apparent Km (~20–30 µM for standard quenched peptides) to keep assay conditions in the linear range where IC50 ≈ Ki. Pre-incubate enzyme with inhibitor for 15 minutes at 25 °C before adding substrate. Include a no-inhibitor control and a metal-chelator positive inhibition control (1 mM EDTA) in each plate. Note that the recombinant lacks the propeptide, so inhibitor mechanisms targeting prodomain interactions will not be detected in this format.

Can I use Meprin A (Recombinant) as a positive control for Western blot with the matched RP-MeprinA antibody?

Yes — this is one of the primary design intentions. RP-MeprinA (anti-Meprin A rabbit polyclonal, /anti-meprin-a-rabbit-polyclonal-antibody) is raised and validated in the same laboratory against the same human MEP1A sequence, giving guaranteed epitope compatibility. Load 10–50 ng of recombinant per lane under reducing conditions alongside your cell or tissue lysate. Expect a band at ~90–100 kDa. This lane serves as both a molecular weight reference and an antibody sensitivity control, and confirms that the RP-MeprinA antibody is working before you interpret endogenous signals.

How much recombinant Meprin A should I load for a Western blot positive control lane, and what exposure time is typical?

10–25 ng per lane is sufficient for a clean, well-resolved band at ~90–100 kDa when using RP-MeprinA at a 1:1,000–1:2,000 dilution and standard HRP-secondary detection. Loading 50 ng gives a stronger signal useful during antibody titration. Exposure time on film or digital imager is typically 30–120 seconds at these loads. Over-loading (>100 ng) can cause smearing due to glycoprotein heterogeneity. Run alongside a broad-range MW marker; the glycoprotein band may appear diffuse rather than a sharp single band — this is expected.

What are the storage and handling requirements for Meprin A (Recombinant), and how long is it stable after thawing?

Store at -20 °C in the supplied single-use aliquots (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10% glycerol). Avoid repeated freeze-thaw cycles — each cycle can reduce metalloproteinase activity by 15–30%. After thawing, keep on ice and use within 4–6 hours; do not refreeze. If same-day use is not possible, the thawed protein can be held at 4 °C for up to 24 hours with acceptable activity retention, but longer storage at 4 °C is not recommended. Purity is >90–95% by SDS-PAGE; endotoxin is <0.1 EU/µg by LAL.