ECE-1 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ECE-1 on SDS-PAGE and Western blot?

ECE-1 (UniProt P42892) has a predicted molecular weight of ~87 kDa based on its 770-amino-acid sequence. Because this recombinant is produced in HEK293 cells, mammalian N-linked glycosylation shifts the apparent molecular weight on SDS-PAGE to approximately 100–110 kDa under denaturing, reducing conditions — consistent with what is observed for native ECE-1 in endothelial cell lysates. If you are running this as a positive control alongside RP-ECE1 antibody blots, load 20–50 ng per lane and expect a diffuse band in the 100–110 kDa region due to glycan heterogeneity.

Which isoform of ECE-1 does this recombinant correspond to, and does it include the transmembrane domain?

This recombinant corresponds to the ECE-1 isoform that retains the catalytically active extracellular metalloprotease domain. The full-length protein is a type II transmembrane zinc metalloprotease; the recombinant construct expresses the ectodomain sufficient for big endothelin-1 cleavage activity, removing the need for membrane solubilization in solution-phase assays. The zinc-binding HEXXH motif and the Trp21–Val22 scissile-bond specificity determinants are intact. If your experiment requires the native membrane topology, this soluble ectodomain form is the standard comparator used in kinetic and inhibitor-screening studies.

What substrate does ECE-1 cleave and how do I set up a fluorogenic activity assay with this recombinant?

ECE-1 (EC 3.4.24.71) cleaves the Trp21–Val22 bond of big endothelin-1 (big ET-1) to generate the 21-residue vasoactive peptide ET-1. For fluorogenic assays, a quenched fluorescent big ET-1 analogue (e.g., Mca-big ET-1 peptide substrates) is the standard choice. Run reactions in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 at 37°C. A starting enzyme concentration of 1–5 nM recombinant ECE-1 with substrate concentrations spanning 1–100 µM allows reliable Km determination. Confirm linearity of signal with time before endpoint reads.

What buffer conditions are optimal for ECE-1 activity assays, and does the storage buffer interfere?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — is directly compatible with standard ECE-1 activity assays. Dilute the stock into the same buffer immediately before use to keep the glycerol contribution below 1% in the final assay volume, as higher glycerol concentrations can modestly suppress kcat. ECE-1 activity is zinc-dependent; avoid chelating agents such as EDTA or EGTA entirely. Phosphoramidon (10 µM) serves as a reliable positive inhibitor control for assay validation. pH 7.5 is optimal; activity drops noticeably below pH 6.5 or above pH 8.5.

How do I determine IC50 values for metalloprotease inhibitors using recombinant ECE-1?

Set up the inhibitor dose-response at a fixed ECE-1 concentration (2–5 nM) and a substrate concentration at or below the Km of big ET-1 (~10–20 µM for most fluorogenic analogues), which maximises sensitivity to competitive inhibitors. Pre-incubate enzyme with inhibitor for 15 minutes at 37°C before initiating the reaction with substrate. Phosphoramidon, a benchmark ECE-1 inhibitor with a reported IC50 in the low-nanomolar range, should anchor every plate as a reference compound. Use at least eight inhibitor concentrations spanning three log units and fit data to a four-parameter logistic model.

Can I use REC-ECE1 as a positive control for Western blot with the matched RP-ECE1 antibody?

Yes — this is one of the primary intended uses of REC-ECE1. The matched antibody RP-ECE1 (rabbit polyclonal; see /anti-ece-1-rabbit-polyclonal-antibody) was validated against HEK293-expressed ECE-1, so these two reagents are guaranteed compatible for Western blot. Load 20–50 ng of REC-ECE1 per lane alongside your cell lysate samples. The recombinant will run at ~100–110 kDa (glycosylated), providing a clean, reproducible reference band. This eliminates the common ambiguity of whether a faint or absent band in experimental lanes reflects low ECE-1 expression or antibody/transfer failure.

How much REC-ECE1 should I load per lane for a Western blot positive control, and what antibody dilution should I use with RP-ECE1?

Load 20–50 ng of REC-ECE1 per lane for a strong, non-saturating positive control band. For the matched RP-ECE1 rabbit polyclonal antibody, a primary dilution of 1:500–1:2,000 in 5% non-fat milk/TBST is a reliable starting point for standard ECL detection on PVDF membranes. Titrate to your specific chemiluminescence system — HRP-based detection with a 1:1,000 primary typically gives a clean band within a 1–5 minute exposure. Since both the recombinant and the antibody originate from the same program, no additional cross-reactivity qualification is needed before use.

How should I store and handle REC-ECE1 to preserve activity, and what is the recommended shelf life?

Store at -20°C in the single-use aliquots supplied. The 10% glycerol in the storage buffer provides cryoprotection; do not dilute to working concentration until immediately before use. Repeated freeze-thaw cycles measurably reduce metalloprotease activity — even two additional cycles can reduce recovered activity by 20–30% for zinc metalloproteases of this class. At -20°C in the original aliquots, activity is stable for at least 12 months. For short-term bench use (within one day), hold on ice; do not leave at room temperature for more than 30 minutes before initiating assays.