Neurotrypsin (Recombinant)

Frequently Asked Questions

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

The unprocessed human Neurotrypsin polypeptide has a predicted molecular weight of ~99 kDa based on its 875-amino-acid sequence (UniProt P56730). Because this recombinant is produced in HEK293 cells with native-like N-linked glycosylation, the apparent molecular weight on reducing SDS-PAGE typically runs between 110–130 kDa — noticeably higher than the sequence-predicted mass. When running a Western blot positive control alongside our matched anti-Neurotrypsin rabbit polyclonal (RP-Neurotrypsin), expect the band in this 110–130 kDa range rather than at the theoretical 99 kDa.

Does recombinant Neurotrypsin undergo autocatalytic or protease-mediated processing, and which form does TPB supply?

Full-length Neurotrypsin is expressed as a single-chain zymogen-like precursor. Physiologically, the C-terminal trypsin-like domain can be released by limited proteolysis, generating an ~80 kDa fragment retaining the SRCR domains and an ~36 kDa catalytic fragment. TPB's REC-Neurotrypsin is supplied as the full-length, largely intact 875-aa form produced in HEK293 cells. If your assay requires the isolated catalytic domain or a specific truncation, you should plan an in-vitro processing step or request the relevant fragment construct. Activity of the full-length form toward synthetic substrates is confirmed prior to release.

What chromogenic or fluorogenic substrate should I use to measure recombinant Neurotrypsin serine protease activity?

Neurotrypsin cleaves after basic residues (Arg/Lys), consistent with its trypsin-like S1 specificity. For activity assays, fluorogenic substrates such as Boc-Phe-Ser-Arg-AMC or tosyl-Gly-Pro-Arg-AMC (t-GPR-AMC) work well as starting points. Chromogenic substrates like Tos-Gly-Pro-Arg-pNA can also be used for plate-reader formats. Run reactions in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20 at 37°C. Include a soybean trypsin inhibitor (SBTI) or PMSF control to confirm serine-protease-dependent signal and rule out contaminating trypsin activity.

What buffer and assay conditions are optimal for Neurotrypsin recombinant protein proteolytic activity experiments?

The storage buffer for REC-Neurotrypsin is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For activity assays, dilute the protein into a reaction buffer of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20 (to minimize surface adsorption), and incubate at 37°C. Glycerol from the storage buffer is typically diluted to ≤1% in the final reaction volume, which does not meaningfully affect activity. Avoid DTT concentrations above 1 mM, as excess reducing agent can disrupt the disulfide bonds critical for correct folding of the SRCR and kringle domains.

What starting concentration of recombinant Neurotrypsin should I use in a proteolytic activity or substrate cleavage assay?

For fluorogenic substrate assays (e.g., Boc-Phe-Ser-Arg-AMC at 50–200 µM), a reasonable starting enzyme concentration is 50–200 nM recombinant Neurotrypsin. Titrate down from there to identify linear-range conditions for your specific substrate and instrument. For cell-based or extracellular matrix degradation experiments, 10–100 nM added exogenously to conditioned medium or a reconstituted matrix is a reported working range in the literature. Always include a no-enzyme blank and a serine protease inhibitor (1 mM PMSF or 10 µM leupeptin) control to confirm signal specificity.

Can I use REC-Neurotrypsin as a positive control for Western blot with the RP-Neurotrypsin rabbit polyclonal antibody?

Yes — that is one of the primary intended uses for this pairing. Load 20–50 ng of REC-Neurotrypsin per lane on a standard 8–10% SDS-PAGE gel; this quantity reliably produces a clean band at ~110–130 kDa with our matched RP-Neurotrypsin antibody. The recombinant and antibody are produced from the same source construct and validated together in-house, so cross-reactivity is confirmed rather than predicted. This makes the pairing useful both as a daily positive control and for establishing antibody working dilutions when you are setting up RP-Neurotrypsin for the first time on your samples.

How much recombinant Neurotrypsin should I load for Western blot positive control alongside cell lysate samples?

Start with 25–50 ng of REC-Neurotrypsin per lane, run adjacent to your cell lysate lanes on the same gel. At this loading, RP-Neurotrypsin (SKU: RP-Neurotrypsin) at a 1:1000–1:2000 primary antibody dilution typically yields a strong, clean band at ~110–130 kDa within a standard 1-hour ECL exposure. If your lysate lanes require longer exposure times due to low endogenous expression, you may reduce the recombinant control load to 10–15 ng to avoid signal saturation on the same blot. Purity of >95% by SDS-PAGE means the dominant band is neurotrypsin, reducing ambiguity.

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

REC-Neurotrypsin is shipped on dry ice and should be stored at −20°C immediately upon receipt in single-use aliquots. Repeated freeze-thaw cycles measurably reduce enzymatic activity due to the structural complexity of the multi-domain architecture (kringle, four SRCR domains, catalytic domain), so aliquot before freezing if your stock arrives as a single vial. On ice, the protein is stable for 4–6 hours during an experiment. Under proper −20°C storage, activity is maintained for at least 12 months from the date of manufacture. Do not store at 4°C long-term; activity loss is significant within days at that temperature.