Anti-Neurotrypsin Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Neurotrypsin on Western blot?

The full-length Neurotrypsin precursor (PRSS12) runs at approximately 97 kDa, corresponding to the 875-amino acid translation product. Because Neurotrypsin undergoes proteolytic processing to generate the active enzyme, you may observe additional bands representing maturation intermediates or the cleaved active form, typically in the 60–70 kDa range depending on cleavage sites and glycosylation state. Brain lysates often show multiple immunoreactive species. Include a positive control such as human brain or spinal cord lysate and confirm band identity by peptide competition or siRNA knockdown if you see unexpected patterns.

What starting dilution should I use for Neurotrypsin Western blot?

We recommend a starting dilution of 1:1000 for Western blot based on validation data. This polyclonal typically yields clean signal in that range when using 20–40 µg of brain or neuronal lysate per lane and standard chemiluminescent detection. If your sample has lower Neurotrypsin expression or you are working with non-neural tissues, begin at 1:500 and titrate upward. Always run a dilution series with your specific lysate and detection system to optimize signal-to-noise, as ECL sensitivity varies between substrates and imaging platforms.

Does this antibody detect Neurotrypsin in non-human primate samples?

This antibody has been validated for human Neurotrypsin and is predicted to cross-react with monkey orthologs based on sequence homology in the immunogen region. Primate PRSS12 sequences share greater than 95 percent identity across key epitope domains. If you are working with cynomolgus, rhesus, or other Old World primates, cross-reactivity is likely but should be confirmed in your hands with a pilot Western blot. For New World monkeys or more distant species, empirical testing is necessary; we have not formally validated those applications.

Which brain regions or tissues express Neurotrypsin at detectable levels?

Neurotrypsin is most abundant in central nervous system tissues, particularly hippocampus, cortex, and spinal cord, where it participates in synaptic remodeling. Expression is developmentally regulated and peaks during periods of circuit refinement, but remains detectable in adult brain regions associated with learning and memory. Peripheral expression is lower; skeletal muscle shows modest signal consistent with its role at the neuromuscular junction. For positive controls, use adult human or mouse whole-brain lysate. Liver, kidney, and non-neural cell lines typically yield little to no signal.

What sample preparation is best for detecting secreted Neurotrypsin?

Neurotrypsin is a secreted serine protease, so it can be detected in conditioned media from neuronal cultures or cerebrospinal fluid in addition to cell and tissue lysates. For culture supernatants, concentrate at least tenfold by ultrafiltration or precipitation (TCA or acetone) to reach detectable levels on Western blot. When preparing tissue lysates, include protease inhibitors targeting serine proteases such as AEBSF or PMSF to prevent autocatalytic degradation. Brain tissue should be homogenized promptly after dissection and kept on ice to preserve immunoreactivity.

Can I use this antibody for immunohistochemistry on brain sections?

Triple Point Biologics antibodies are validated for Western blot and immunohistochemistry as part of our standard workflow. This rabbit polyclonal should be suitable for IHC on formalin-fixed paraffin-embedded or frozen brain sections. For FFPE, antigen retrieval is typically required; citrate buffer (pH 6.0) heat-induced epitope retrieval is a reasonable starting point. Begin antibody dilution at 1:200 to 1:500 and titrate based on signal intensity and background. Positive control sections from hippocampus or cortex will help optimize staining conditions.

How should I store this Neurotrypsin antibody for long-term stability?

Store the antibody at –20°C as supplied; avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. For routine use, aliquot upon receipt into single-use volumes (10–20 µL) and keep working aliquots at 4°C for up to one month. Do not store diluted antibody in blocking buffer for more than a few days, as bacterial growth and protein degradation can compromise performance. If you observe unusual background or loss of signal over time, thaw a fresh aliquot and compare side-by-side to assess whether freeze-thaw damage has occurred.

What controls should I include when detecting Neurotrypsin by Western blot?

Include a positive control lysate from human or rodent brain tissue known to express Neurotrypsin at high levels, such as hippocampus or cortex. A negative control using a non-neural cell line or tissue (HEK293, HeLa, or liver) helps confirm specificity. If you see unexpected bands, consider peptide competition with the immunogen or an siRNA/shRNA knockdown in a neuronal cell model to verify that the signal is PRSS12-specific. Running a no-primary-antibody lane rules out secondary-antibody artifacts.