Anti-Nardilysin Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Nardilysin migrates at approximately 130 kDa on SDS-PAGE, consistent with its 1151-residue sequence (predicted MW ~127 kDa). You may observe additional bands around 110-120 kDa representing proteolytic fragments or alternatively processed forms, particularly in mitochondrial-enriched fractions. Some reports describe a cleaved cytosolic form that lacks the N-terminal mitochondrial targeting sequence. If you see multiple bands, consider whether your sample preparation retained full-length protein; harsh lysis conditions or freeze-thaw cycles can promote cleavage. Running a parallel lane with recombinant nardilysin or a well-characterized positive-control lysate helps confirm identity.

What starting dilution should I use for nardilysin Western blots?

Start at 1:1000 dilution in blocking buffer containing 5 percent non-fat milk or BSA. Nardilysin is moderately abundant in brain, heart, and metabolically active tissues but lower in many cell lines, so you may need to optimize between 1:500 and 1:2000 depending on your sample. Incubate overnight at four degrees Celsius for best signal-to-noise. Load 20-40 micrograms total protein per lane; mitochondrial-enriched fractions typically yield stronger signals than whole-cell lysates. If background is high at 1:1000, increase dilution and extend exposure time rather than reducing antibody concentration too quickly.

Does nardilysin localize to mitochondria or cytosol, and how does that affect sample prep?

Nardilysin is primarily mitochondrial but detectable in cytosolic fractions and secreted forms depending on cell type and metabolic state. For strongest signal, use mitochondrial-enrichment protocols or whole-cell RIPA lysis with protease inhibitors. Avoid hypotonic buffers that may rupture mitochondria incompletely, leaving nardilysin in pellet fractions. If studying ectodomain shedding or neuregulin activation, include conditioned media or membrane preparations. Subcellular fractionation can clarify whether you are detecting full-length mitochondrial nardilysin or cleaved cytosolic isoforms. Always include a mitochondrial marker like VDAC or COX IV to verify fraction purity.

Is this nardilysin antibody cross-reactive with rat and mouse samples?

Human nardilysin is validated; rat and mouse cross-reactivity are predicted based on sequence homology exceeding 85 percent in the immunogen region. Researchers have successfully used rabbit polyclonal nardilysin antibodies on rodent brain and heart lysates, but we recommend titrating dilution and running a positive control the first time. Expect similar banding patterns around 130 kDa. If signal is weak in mouse samples, consider enriching mitochondria or increasing protein load to 40 micrograms per lane. Canine cross-reactivity is also predicted but less commonly reported; empirical validation in your tissue of interest is prudent.

What are good positive and negative controls for nardilysin Western blots?

Brain, heart, and liver lysates serve as strong positive controls because nardilysin is expressed at moderate to high levels in metabolically active tissues. Neuronal cell lines like SH-SY5Y or primary cortical cultures also work well, especially if you are studying neuregulin signaling. HEK293 or COS cells transfected with nardilysin expression constructs provide an unambiguous positive band. For negative controls, consider NRDC-knockout cell lines if available, or tissues with very low endogenous expression. Recombinant nardilysin protein, when run alongside lysates, confirms antibody specificity and helps distinguish full-length from cleaved forms.

Can I use this antibody for immunofluorescence or immunohistochemistry on nardilysin?

Triple Point Biologics antibodies are routinely validated for immunohistochemistry and immunofluorescence alongside Western blot. For IF, try 1:100 to 1:200 dilution; nardilysin should show punctate mitochondrial staining that co-localizes with MitoTracker or TOM20. Antigen retrieval—citrate buffer, pH six, ten minutes at sub-boiling—improves IHC signal in formalin-fixed paraffin sections, particularly in brain and cardiac tissue. Expect cytoplasmic and perinuclear signal. Methanol fixation often works better than paraformaldehyde for preserving mitochondrial epitopes in cultured cells. Include a no-primary-antibody control to assess non-specific secondary binding.

How should I store the nardilysin antibody and how long is it stable?

Store at minus twenty degrees Celsius in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100-microgram pack size is convenient for aliquoting 10-20 microliter volumes in working dilution or as concentrated stock. Antibody remains stable for at least twelve months under these conditions. For short-term use within two to four weeks, you may hold aliquots at four degrees Celsius in the presence of 0.02 percent sodium azide or commercial stabilizer; however, freezing is preferable for long-term preservation. Do not store in frost-free freezers that undergo temperature cycling.

Does nardilysin have isoforms or splice variants I should be aware of?

The canonical nardilysin transcript encodes the 1151-residue isoform. Alternative splicing has been reported but is not well characterized in most tissues. You may detect proteolytic cleavage products, particularly a truncated form missing the N-terminal mitochondrial import sequence that appears in cytosolic fractions. These fragments retain catalytic activity and are biologically relevant. On Western blots, minor bands below 130 kDa likely represent such cleavage events rather than distinct splice isoforms. If you are comparing tissues or disease models, expect variation in the ratio of full-length to cleaved nardilysin, which can reflect differences in mitochondrial integrity or proteolytic regulation.