Meprin B (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Meprin B run at on SDS-PAGE and Western blot?

Meprin B (REC-MeprinB) is expressed from the full 701-amino-acid human sequence (UniProt Q16820), giving a predicted polypeptide mass of ~79 kDa. Because it is produced in HEK293 cells and carries native-like N-glycosylation, the apparent molecular weight on reducing SDS-PAGE typically runs between 90–110 kDa. The exact migration shifts slightly depending on glycosylation load in your cell line context. When using this preparation as a Western blot positive control alongside our matched antibody RP-MeprinB, load 50–100 ng per lane and expect the broad, diffuse band characteristic of heavily glycosylated metalloendopeptidases.

Is this recombinant Meprin B the full-length transmembrane form or the shed ectodomain?

REC-MeprinB is the soluble ectodomain form — the extracellular portion that retains the prodomain (removed during activation), the astacin catalytic domain, and the flanking MAM, TRAF, and EGF-like modules, but lacks the single-pass transmembrane anchor and cytoplasmic tail. This matches the shed form generated in vivo by ADAM10 cleavage. It is supplied as an active enzyme in solution, meaning the prodomain has been processed to yield the mature, catalytically competent species. If your experiment requires the membrane-tethered topology, this preparation is not a substitute.

What substrates does Meprin B cleave and how do I set up a fluorogenic activity assay?

Meprin B exhibits strong preference for acidic residues at the P1′ position. For fluorogenic activity assays, the peptide substrate Mca-YVADAPK(Dnp)-OH (containing an Asp at P1′) is commonly used and provides a reliable signal-to-noise ratio. Assay conditions: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, 10 µM substrate, and 1–5 nM REC-MeprinB at 37°C. Monitor fluorescence at Ex/Em 320/405 nm. Biologically characterized substrates include procollagen I/III, E-cadherin, IL-1β, IL-18, FGF19, VEGFA, KLK7, ADAM10, and amyloid precursor protein — useful for designing cleavage validation experiments.

What buffer should I use for Meprin B activity assays and is EDTA compatible?

REC-MeprinB is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. For activity assays, dilute directly into a matched assay buffer (same formulation, minus glycerol) to avoid carry-over inhibition. EDTA is incompatible — as a zinc-dependent astacin metalloendopeptidase, chelation of the active-site zinc by EDTA causes rapid, irreversible inactivation. Similarly, avoid 1,10-phenanthroline, DTT above 1 mM (which can reduce structural disulfides), and detergents above 0.01% SDS. If your downstream assay requires a reducing environment, test DTT tolerance empirically starting at 0.1 mM.

What starting concentration of recombinant Meprin B should I use for inhibitor IC50 measurements?

For inhibitor IC50 experiments, use REC-MeprinB at 1–5 nM in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 with 10 µM fluorogenic substrate. Keep enzyme concentration at least 10-fold below the inhibitor's anticipated Ki to satisfy the Morrison tight-binding correction criteria. Pre-incubate enzyme with inhibitor for 30 minutes at room temperature before initiating the reaction with substrate. Ensure DMSO from inhibitor stocks does not exceed 0.5% v/v, as higher concentrations measurably reduce Meprin B activity in our internal benchmarking. Run at least three independent replicates across an 8–10 point inhibitor dilution series.

How much recombinant Meprin B should I load as a Western blot positive control for the RP-MeprinB antibody?

For use as a positive control with our matched rabbit polyclonal RP-MeprinB (/anti-meprin-b-rabbit-polyclonal-antibody), load 50–100 ng of REC-MeprinB per lane under reducing conditions. At this loading, RP-MeprinB typically detects a band at 90–110 kDa (reflecting glycosylation). This antibody-recombinant pair was developed in the same lab, so antigen compatibility is confirmed rather than predicted. If you are running cell or tissue lysates alongside, the recombinant lane provides a reliable size marker for the endogenous band and confirms antibody performance in the same blot without ambiguity.

Can I use REC-MeprinB to validate the RP-MeprinB antibody for IHC or immunoprecipitation?

REC-MeprinB is most directly useful for validating RP-MeprinB in Western blot, where you can confirm antigen recognition under denaturing conditions. For immunoprecipitation validation, the soluble recombinant ectodomain can be spiked into lysis buffer to confirm pull-down efficiency by RP-MeprinB before moving to endogenous targets. IHC validation requires a cell-based or tissue section context; the recombinant itself is not suitable as a direct IHC control, but RP-MeprinB has validated IHC reactivity (see antibody product page). Cross-reactivity with mouse and rat Meprin B is predicted based on sequence homology but should be confirmed empirically in your system.

How should I store and handle recombinant Meprin B to preserve enzymatic activity long-term?

REC-MeprinB is supplied in single-use aliquots in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 and should be stored at -20°C. Avoid repeated freeze-thaw cycles — each cycle measurably reduces metalloendopeptidase activity. Upon first thaw, keep on ice and use within the same working session. For dilutions into assay buffer, prepare fresh working stocks at ≥10 ng/µL to minimize surface-adsorption losses; use low-binding tubes. Do not store diluted enzyme at 4°C for more than 4 hours. Purity is >90% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL, so no additional clean-up is required before cell-based assays.