ECE-2 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ECE-2 on SDS-PAGE and Western blot?

ECE-2 (UniProt O60344) has a predicted polypeptide MW of ~86 kDa, but the HEK293-expressed recombinant runs at approximately 100–110 kDa under denaturing conditions due to N-linked glycosylation. This glycoform closely mirrors endogenous ECE-2 observed in human brain and placental tissue lysates. When running SDS-PAGE for purity assessment, expect a dominant band in the 100–110 kDa region at >90% purity. If you observe a doublet in this range, this is consistent with differential glycosylation and does not indicate degradation.

What processing form of ECE-2 does this recombinant represent — full-length or ectodomain?

REC-ECE2 represents the catalytically active ectodomain of human ECE-2, comprising the extracellular zinc metallopeptidase domain with the transmembrane anchor absent. This soluble ectodomain format retains the zinc-binding HEXXH motif and disulfide architecture required for activity. It is not the full-length type II integral membrane protein, so it is not suitable for membrane topology or full-length overexpression studies. For enzymatic cleavage assays and antibody validation experiments, the ectodomain form is the functionally relevant preparation.

What substrates does recombinant ECE-2 cleave and what fluorogenic substrate should I use for activity assays?

ECE-2 (EC 3.4.24.71) efficiently cleaves big endothelin-1 at the Trp21–Val22 bond to generate endothelin-1 (ET-1), and also processes big ET-2 and big ET-3. For fluorogenic assays, McaBk2 (Mca-RPPGFSAFK(Dnp)-OH), a bradykinin-based substrate validated for M13-family metallopeptidases, is widely used and gives robust signal at low enzyme concentrations. Substrate concentration of 10–20 µM with 1–5 nM recombinant enzyme in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2) at 37°C provides a linear-range readout suitable for IC50 determination.

What buffer conditions are optimal for recombinant ECE-2 enzymatic activity assays?

REC-ECE2 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — a formulation that stabilises the zinc metallopeptidase fold. For activity assays, dilute directly into the same buffer minus glycerol to avoid viscosity effects on fluorescence readings. ECE-2 activity is optimal between pH 5.5–6.8 (lysosomal conditions) in some cellular contexts, but the recombinant ectodomain shows measurable activity at pH 7.0–7.5 in vitro. Include 0.01% BSA as carrier when working at sub-nanomolar enzyme concentrations to prevent surface adsorption losses.

What starting concentration of recombinant ECE-2 should I use for a peptide cleavage or inhibitor IC50 assay?

For fluorogenic McaBk2 substrate assays, a starting enzyme concentration of 2–5 nM REC-ECE2 is recommended at 37°C for a 60-minute endpoint or continuous kinetic read. For HPLC-based big endothelin-1 cleavage assays, 10–20 nM enzyme with 1–5 µM substrate gives sufficient product for UV detection. When running inhibitor IC50 experiments, keep enzyme concentration at or below the Ki of your tightest expected inhibitor (typically ≤10 nM) to avoid stoichiometric titration artefacts. Perform a linearity-of-signal check across 1–50 nM enzyme before committing to a full inhibitor panel.

Can I use REC-ECE2 as a positive control for Western blot with the matched ECE-2 antibody RP-ECE2?

Yes — REC-ECE2 and RP-ECE2 are produced and validated in the same laboratory specifically for this use case. Load 20–50 ng of REC-ECE2 per lane alongside your cell or tissue lysate. The anti-ECE-2 rabbit polyclonal (RP-ECE2) detects the recombinant at the expected 100–110 kDa glycoprotein band, providing an unambiguous size marker for endogenous ECE-2 identification. This pairing is also useful for antibody lot-to-lot consistency checks. See the RP-ECE2 product page (/anti-ece-2-rabbit-polyclonal-antibody) for recommended primary antibody dilutions and validated secondary antibody conditions.

How much recombinant ECE-2 should I load for Western blot positive control and what band should I expect?

Load 20–50 ng of REC-ECE2 per lane for a clean, well-resolved positive control band when using RP-ECE2 as the primary antibody. Expect a band at approximately 100–110 kDa reflecting the glycosylated ectodomain. At 50 ng, the band is clearly visible after standard ECL detection with 1–2 minute exposure; loading >100 ng risks blooming that obscures adjacent lysate lanes. If you are comparing against endogenous ECE-2 in HUVEC or brain tissue lysates, the recombinant and endogenous bands should co-migrate, confirming antibody specificity.

How should I store and handle recombinant ECE-2 to maintain activity, and what is the shelf life?

REC-ECE2 is shipped on dry ice and should be stored at -20°C upon receipt in single-use aliquots to avoid repeated freeze-thaw cycles, which progressively reduce metallopeptidase activity. At -20°C, activity is stable for at least 12 months from the date of manufacture. Once thawed, keep on ice and use within 4–8 hours; do not refreeze. For dilutions below 10 nM, prepare working stocks in assay buffer supplemented with 0.01% BSA immediately before use. Do not store diluted enzyme overnight — the zinc metallopeptidase active site is sensitive to prolonged dilute-solution incubation.