Anti-Neprilysin-2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Neprilysin-2 on a Western blot?

Neprilysin-2 (MMEL1) runs at approximately 85-90 kDa on reducing SDS-PAGE, consistent with its 779-residue sequence plus glycosylation. The protein is a type-II membrane metalloprotease with predicted N-glycosylation sites, so the observed band may appear slightly higher than the calculated mass of approximately 87 kDa. Some labs report a diffuse band or doublet due to variable glycosylation states. If you see multiple bands, treatment with PNGase F can help confirm glycosylation. Start with the recommended 1:1000 dilution and expose for 1-5 minutes on ECL.

Does this antibody cross-react with classical neprilysin or CD10?

Neprilysin-2 shares roughly 40 percent sequence identity with classical neprilysin (MME/CD10), both within the M13 peptidase family. While the epitope used to generate this polyclonal has not been disclosed in detail, cross-reactivity with CD10 is possible given structural homology. If your tissue or lysate co-expresses both proteases, run parallel samples with a validated CD10 antibody or use tissue known to selectively express one isoform. Testis lysates are ideal for Neprilysin-2 since expression is highly enriched there, whereas kidney and lymphoid tissues predominantly express CD10.

Which human tissues express Neprilysin-2 at detectable levels for Western blot?

Neprilysin-2 expression is most abundant in testis and male reproductive tract tissues, making human testis lysate the positive control of choice. Lower expression has been reported in prostate and epididymis. Expression in other tissues is minimal or absent by most transcriptomic surveys. If you are working with non-reproductive tissues, consider a longer exposure or enrichment step, though signal may still be undetectable. For cell lines, consult RNA-seq databases; expression is rare outside germline-derived or testicular carcinoma lines. Kidney or spleen lysates serve as useful negative controls.

What starting dilution should I use for immunohistochemistry with this antibody?

this antibody is validated for Western blot; IHC validation is in progress, and a starting dilution of 1:100 to 1:200 is typical for paraffin-embedded sections. Antigen retrieval is usually required; citrate buffer (pH 6.0) at sub-boiling temperature for 20 minutes works well for many membrane metalloproteases. Testis sections are the recommended positive control. Block with normal goat serum if using a goat anti-rabbit secondary. Optimize incubation time and dilution with your specific tissue and detection system; some labs achieve clean signal at 1:400 after overnight incubation at four degrees Celsius.

Is this Neprilysin-2 antibody likely to work in non-human primate or dog samples?

The antibody is validated in human samples and shows predicted cross-reactivity with monkey and dog based on sequence homology in the likely epitope region. Cynomolgus and rhesus macaque Neprilysin-2 share greater than 90 percent identity with human, so cross-reactivity is probable. Canine ortholog identity is slightly lower but still substantial. For these species, start at the same 1:1000 dilution for Western blot and confirm with a positive control lysate from testis if available. Mouse and rat orthologs diverge more; cross-reactivity is not predicted and has not been validated.

How should I prepare cell lysates to preserve Neprilysin-2 for Western blot?

Neprilysin-2 is a membrane-bound metalloprotease, so include detergent in your lysis buffer. RIPA buffer (150 mM NaCl, 1 percent NP-40, 0.5 percent deoxycholate, 0.1 percent SDS) efficiently solubilizes membrane proteins and is compatible with downstream Western blot. Add protease inhibitors immediately before lysis; EDTA-free cocktails are preferable since MMEL1 is a zinc metalloprotease and you may wish to preserve activity for parallel assays. Sonication or passage through a narrow-gauge needle can improve yield. Avoid repeated freeze-thaw cycles; aliquot lysates and store at minus 80 degrees Celsius.

What is the recommended storage condition and shelf life for this antibody?

Store the antibody at minus 20 degrees Celsius as supplied. For routine use, a working aliquot can be kept at four degrees Celsius for up to one month with sodium azide (0.02 to 0.05 percent final) to prevent microbial growth. Avoid repeated freeze-thaw cycles, which denature IgG and reduce titer; divide into single-use aliquots upon receipt if you use the antibody infrequently. Frozen aliquots stored at minus 20 degrees typically retain activity for at least one year. Some labs store long-term stock at minus 80 degrees for extended stability beyond two years.

Can I use this antibody for immunofluorescence on live or fixed cells?

this antibody is validated for Western blot; immunofluorescence validation is in progress on fixed cells. For live-cell staining, Neprilysin-2 is a type-II membrane protein with its catalytic domain exposed extracellularly, so the epitope accessibility depends on whether the immunogen corresponds to the ectodomain. Most commercial antibodies target intracellular or membrane-spanning regions and require permeabilization. Fix cells with four percent paraformaldehyde for ten minutes, permeabilize with 0.1 to 0.3 percent Triton X-100, and start with a 1:50 to 1:100 dilution. Optimize on a known Neprilysin-2-positive cell line if available.