Neuroserpin (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Neuroserpin run at on SDS-PAGE or Western blot?

The predicted molecular weight of Neuroserpin (SERPINI1; Q99574) from its 410-amino acid sequence is approximately 46 kDa. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation adds roughly 5–10 kDa of apparent mass, so expect a diffuse band migrating between 50–60 kDa under reducing SDS-PAGE conditions. The glycosylation pattern is consistent with the native secreted form, so this migration closely mirrors what you would see in conditioned media or brain tissue lysates. Purity is >95% by SDS-PAGE, so a single dominant band is expected.

Is this recombinant Neuroserpin the full-length mature secreted form or does it include the signal peptide?

The recombinant protein corresponds to the mature secreted form of human Neuroserpin (SERPINI1), expressed in HEK293 cells with the native signal peptide directing secretion and subsequent removal during processing. The final purified product therefore lacks the signal peptide and represents the circulating, inhibitory-competent serpin. This is the biologically relevant conformation for inhibitory activity assays and is the same form detected by the matched antibody RP-Neuroserpin (/anti-neuroserpin-rabbit-polyclonal-antibody) on Western blot of brain or plasma samples.

Which serine proteases does recombinant Neuroserpin inhibit, and what is its inhibitory selectivity profile?

Neuroserpin operates via a suicide-substrate (covalent acyl-intermediate) mechanism and forms stable 1:1 stoichiometric complexes with its principal targets: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). It also inhibits plasmin, though with lower efficiency. Importantly, it shows negligible inhibitory activity against thrombin, distinguishing it from broader-spectrum serpins such as PAI-1. This selectivity makes it the appropriate recombinant for dissecting tPA/uPA-dependent signalling in neuroplasticity, ischemia, or tumor invasion studies without confounding thrombin-pathway interference.

What substrate and assay format should I use to measure recombinant Neuroserpin inhibitory activity against tPA?

A standard second-order inhibition assay uses a chromogenic or fluorogenic plasminogen activator substrate — commonly Boc-Glu-Gly-Arg-AMC or Spectrozyme tPA (H-D-Ile-Pro-Arg-pNA) — in a continuous kinetics format. Pre-incubate Neuroserpin with tPA (both at 50–200 nM) in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20, 37 °C, then add substrate and monitor residual tPA activity over 30–60 minutes. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is directly compatible; simply dilute to working concentration to keep glycerol below 1% in the final reaction.

What starting concentration of recombinant Neuroserpin should I use for IC50 or stoichiometry of inhibition experiments?

For stoichiometry-of-inhibition (SI) titrations, use fixed tPA at 10–50 nM and titrate Neuroserpin across a 0.5–5× molar ratio range. Because Neuroserpin is a mechanism-based inhibitor, SI values (typically 1.5–3 for tPA) matter more than a classical IC50. For initial activity checks, 100–200 nM Neuroserpin against 50 nM tPA in assay buffer at 37 °C gives robust inhibition within 30 minutes. Avoid using concentrations below 10 nM without carrier protein (0.1% BSA), as surface adsorption losses become significant at low picomolar ranges.

How should I store and handle recombinant Neuroserpin to preserve inhibitory activity, and what is its shelf life?

Upon receipt, store at −20 °C in single-use aliquots. The supplied storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) stabilises the native serpin conformation; do not remove glycerol by dialysis unless your assay strictly requires it, as this accelerates latency transition. Repeated freeze-thaw cycles promote polymerisation — a known artefact with serpins — which abolishes inhibitory activity without necessarily altering SDS-PAGE band appearance. When handled correctly, activity is stable for at least 12 months at −20 °C. Thawed aliquots can be kept at 4 °C for up to 48 hours.

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

Yes — this recombinant is the validated positive control for the matched antibody RP-Neuroserpin (/anti-neuroserpin-rabbit-polyclonal-antibody). Load 20–50 ng per lane alongside your tissue lysate. Because the HEK293-expressed protein carries native-like glycosylation, the recombinant band migrates at 50–60 kDa — essentially co-migrating with the endogenous band from brain or conditioned media samples — which confirms antibody specificity without ambiguity from molecular weight discrepancy. Running a titration of 10, 25, and 50 ng also lets you estimate relative Neuroserpin abundance in your sample by densitometry.

How much recombinant Neuroserpin should I load to optimise Western blot sensitivity when validating RP-Neuroserpin antibody?

For antibody validation and sensitivity assessment with RP-Neuroserpin, a three-point loading series of 10 ng, 25 ng, and 50 ng per lane is recommended under reducing conditions. The >95% pure preparation ensures the signal reflects Neuroserpin specifically, not background contaminants. At 25 ng on a standard PVDF membrane, a clean band at 50–60 kDa should be detectable with RP-Neuroserpin at 1:1000–1:2000 dilution using standard HRP-secondary detection. If you observe a smear rather than a discrete band, consider whether your sample preparation temperatures promoted serpin polymerisation prior to loading.