Neurolysin (Recombinant)

Frequently Asked Questions

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

The full-length human Neurolysin (UniProt Q9BYT8) is 704 amino acids, giving a predicted molecular weight of approximately 78 kDa. On reducing SDS-PAGE, this HEK293-expressed recombinant typically resolves as a single band at ~78–80 kDa. Minor upward shift relative to the calculated MW is common due to glycosylation or incomplete denaturation of the zinc-coordinated tertiary structure. Purity is >90% by SDS-PAGE. When using this recombinant as a Western blot positive control alongside RP-Neurolysin, expect the primary band at ~78–80 kDa.

Is this recombinant Neurolysin the full-length sequence or a processed mitochondrial form?

This recombinant is produced from the full-length 704-amino-acid human NLN sequence in HEK293 cells. Native Neurolysin contains an N-terminal mitochondrial targeting sequence (approximately residues 1–29) that is cleaved upon mitochondrial import in vivo. The HEK293 expression system may produce a mixture of processed and unprocessed forms depending on construct design — check the certificate of analysis for the specific construct boundaries used in your lot. If isoform resolution is critical for your experiment, the matched antibody RP-Neurolysin can help confirm the processed band by Western blot.

What substrates does recombinant Neurolysin cleave and which is best for a fluorogenic activity assay?

Neurolysin (EC 3.4.24.16) cleaves oligopeptides including neurotensin, bradykinin, dynorphin A, and hemopressin. For fluorogenic peptide cleavage assays, internally quenched fluorescent neurotensin analogs (e.g., Mca-Pro-Leu-Gly-Pro-D-Lys(Dnp)-OH) are the most widely used substrates and give robust signal-to-noise ratios. Substrate concentrations of 10–50 µM in assay buffer are a practical starting range. Hemopressin is the preferred substrate when studying cannabinoid receptor CNR1 signalling contexts, as Neurolysin's degradation of this endocannabinoid ligand is the relevant physiological endpoint.

What buffer conditions are recommended for Neurolysin activity assays with this recombinant?

The storage buffer for this recombinant is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — the CaCl2 inclusion is standard for TPB metalloproteinase preparations to support zinc coordination. For activity assays, a working buffer of 50 mM HEPES or Tris-HCl pH 7.4, 150 mM NaCl, 1 mM ZnCl2 is compatible with published Neurolysin kinetic studies. Avoid strong chelators such as EDTA or EGTA, which strip the catalytic zinc and abolish activity. DTT at concentrations above 1 mM can also interfere; keep reducing agents minimal in the assay well.

What starting enzyme concentration should I use for a Neurolysin kinetic or inhibitor IC50 assay?

For initial kinetic characterisation, 1–10 nM recombinant Neurolysin in the assay well is a reasonable starting range with fluorogenic neurotensin substrates at 10–50 µM. For inhibitor IC50 determinations, use the lowest enzyme concentration that gives a reliable linear signal over your measurement window — typically 2–5 nM — to avoid substrate depletion artifacts and to ensure you are measuring true inhibitor potency rather than stoichiometric titration of active sites. Titrate the enzyme against your specific substrate lot before committing to a full inhibitor screen, as activity can vary between preparations.

Can I use recombinant Neurolysin as a positive control for Western blot with the RP-Neurolysin antibody?

Yes — this recombinant is the recommended positive control for the matched antibody RP-Neurolysin (/anti-neurolysin-rabbit-polyclonal-antibody). Both are produced in the same lab, so epitope compatibility is confirmed rather than inferred. Load 20–50 ng of recombinant Neurolysin per lane on a 10% SDS-PAGE gel alongside your cell lysates. The rabbit polyclonal detects the ~78–80 kDa band under standard reducing conditions. This pairing is particularly useful for antibody validation experiments and for confirming knockdown or overexpression in NLN-edited cell lines, where you need an unambiguous reference band.

How much recombinant Neurolysin should I load for a Western blot positive control lane?

20–50 ng per lane is the standard loading range for a clean, non-saturating signal with RP-Neurolysin on a standard chemiluminescent Western blot. At 50 ng, the ~78–80 kDa band is clearly visible without blooming into adjacent lanes. If you are running a quantitative Western or using near-infrared fluorescent secondary detection, start at 10–20 ng to stay within the linear dynamic range. Dilute the recombinant directly into 1× Laemmli sample buffer immediately before loading; do not pre-dilute in water, as diluting below the glycerol-buffered formulation can affect band sharpness.

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

Store at −20°C in the single-use aliquots as supplied. The formulation — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — is designed for long-term stability at this temperature. Repeated freeze-thaw cycles progressively reduce metalloendopeptidase activity through zinc loss and aggregation; once an aliquot is thawed, use it the same day. If you need working dilutions, prepare them in assay-compatible buffer containing 0.1% BSA to reduce adsorptive losses at low protein concentrations (below ~50 ng/µL). Avoid metal-chelating plasticware additives and ensure all tips and tubes are low-binding grade.