Anti-Neurolysin Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Neurolysin on a Western blot?

Neurolysin migrates at approximately 80 kDa on SDS-PAGE, corresponding to its 704-amino acid sequence (predicted MW ~78 kDa). You may observe minor variation depending on post-translational modifications or gel conditions. The enzyme exists primarily as a single isoform in human tissue, though some groups have reported proteolytic fragments in specific mitochondrial preparations. If you see multiple bands, consider whether your lysis method preserved mitochondrial integrity or inadvertently activated endogenous proteases. Brain and kidney lysates typically yield the cleanest signals given Neurolysin's abundance in those tissues.

Which starting dilution should I use for Western blot with this Neurolysin antibody?

Start at 1:1000 in 5% non-fat milk or BSA in TRIS-buffered saline with Tween-20. This dilution has been validated for human samples and generally provides good signal-to-noise with overnight incubation at 4°C. If you are working with tissue known to express Neurolysin at lower levels, you may trial 1:500. Conversely, mitochondrial-enriched fractions from brain or kidney may permit dilution to 1:2000. Always include a positive control lysate from a high-expression tissue to confirm antibody performance and optimize exposure time before troubleshooting sample-specific issues.

Does this antibody work for mouse and rat Neurolysin, or only human?

The antibody is validated for human Neurolysin and predicted to cross-react with mouse and rat based on sequence homology in the immunogen region. Mouse NLN shares approximately 85% identity with human, and rat is similarly conserved. We have not formally validated reactivity in these species, so if you are working with rodent samples, plan to include a known positive control and be prepared to optimize dilution. Primate, dog, and pig orthologs are also predicted to cross-react. If cross-species work is critical, consider running a small pilot blot before committing to a full experiment.

What is the best sample preparation method to detect mitochondrial Neurolysin?

Because Neurolysin localizes to the mitochondrial intermembrane space, use a lysis buffer that preserves organellar integrity or perform subcellular fractionation. RIPA buffer works for whole-cell lysates, but you will detect Neurolysin alongside cytosolic contaminants. For cleaner detection, isolate mitochondria by differential centrifugation, then lyse the mitochondrial pellet with a mild detergent or freeze-thaw to release intermembrane-space proteins. Include protease inhibitors throughout to prevent degradation. Expect stronger signal in brain, kidney, and liver compared to other tissues. Avoid boiling samples excessively, as this can aggregate mitochondrial proteins and reduce transfer efficiency.

Can I use this antibody for immunofluorescence to visualize Neurolysin in cells?

Yes, Triple Point Biologics antibodies are validated for immunofluorescence applications. For Neurolysin, expect punctate mitochondrial staining in the cytoplasm. Fix cells with 4% paraformaldehyde, permeabilize with 0.1–0.3% Triton X-100, and start with a 1:100 to 1:200 dilution of primary antibody. Co-staining with a mitochondrial marker such as Tom20 or cytochrome c can confirm localization to the intermembrane space. Because the antigen is intracellular, permeabilization is essential. If background is high, increase blocking time or try a more stringent wash protocol. Neuronal and kidney cell lines typically show robust expression.

What positive and negative controls should I include when using this Neurolysin antibody?

Use human brain or kidney lysate as a positive control, since Neurolysin is abundantly expressed in both tissues. HEK293 cells also express moderate levels and are convenient if tissue lysates are unavailable. For a negative control, consider a Neurolysin knockout or knockdown cell line if accessible, or pre-incubate the antibody with the immunizing peptide to demonstrate specificity through signal blockade. Additionally, compare mitochondrial and cytosolic fractions; Neurolysin should enrich in the mitochondrial fraction. Including a loading control such as beta-actin or VDAC helps normalize mitochondrial yield and confirms equal protein loading across lanes.

How should I store this Neurolysin antibody to maintain 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 the stock into single-use volumes immediately upon receipt. If you use the antibody frequently, a working aliquot can be kept at 4°C with 0.02–0.05% sodium azide for up to one month, though –20°C storage is preferable for maximum longevity. Do not store diluted antibody in blocking buffer for more than a few days. Rabbit polyclonal antibodies from Triple Point Biologics have been stable under these conditions for several years when handled properly.

Why am I seeing multiple bands on my Neurolysin Western blot?

If you observe bands in addition to the expected 80 kDa species, first confirm that your positive control shows a single band at the correct size. Multiple bands may arise from incomplete protease inhibition during lysis, leading to Neurolysin degradation. Mitochondrial preparations are especially prone to proteolytic cleavage if samples are not kept cold and processed quickly. Cross-reactivity with other metalloproteases is unlikely given the immunogen design, but cannot be entirely excluded. Non-specific binding may appear if primary antibody concentration is too high; try increasing the dilution. Finally, check for overloading, which can produce artifactual smearing or reveal low-abundance degradation products.