Anti-Insulysin Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Insulysin (IDE) runs at approximately 110-118 kDa on SDS-PAGE, consistent with its 1019-amino acid sequence and predicted mass of 118 kDa. The enzyme is typically detected as a single band under reducing conditions. You may occasionally observe slower-migrating species if post-translational modifications are present or if the enzyme forms SDS-resistant oligomers. For best resolution, use 7.5-10 percent polyacrylamide gels and transfer to PVDF rather than nitrocellulose. The rabbit polyclonal antibody is validated at 1:1000 dilution in human lysates; start there and titrate if signal is weak in your system.

Does this Insulysin antibody cross-react with mouse and rat samples?

The antibody is validated for human Insulysin and predicted to cross-react with mouse and rat based on sequence homology in the immunogen region. Human and mouse IDE share approximately 89 percent identity; rat is similar. Cross-reactivity with primate (monkey), canine, and porcine samples is also predicted but not experimentally validated by Triple Point Biologics. If you are working with non-human species, we recommend running a positive control lysate from the target species alongside human samples to confirm detection. Expect the same approximately 118 kDa band across mammalian species.

Which cell lines or tissues express high levels of Insulysin for positive controls?

Insulysin is broadly expressed, with highest levels in insulin-responsive tissues. Liver, skeletal muscle, kidney, and pancreatic beta-cells are excellent positive-control tissues. Among cultured cell lines, HepG2 (hepatoma), HEK293, and skeletal-muscle-derived myoblasts typically show robust IDE expression. Neuronal cells such as SH-SY5Y also express IDE given its role in amyloid-beta degradation. For negative controls, IDE knockout cell lines are ideal but rare; alternatively, preincubate the antibody with blocking peptide if available, or compare signal intensity across tissues known to vary in IDE expression such as adipose versus liver.

What is the recommended starting dilution for Western blot with this antibody?

Start at 1:1000 dilution in blocking buffer (5 percent nonfat milk or 3 percent BSA in TBST) for Western blot of whole-cell or tissue lysates. This dilution is validated for human samples and typically yields clean signal with 20-40 micrograms total protein per lane. If background is high, increase dilution to 1:2000; if signal is weak in low-expression samples, try 1:500. Incubate overnight at 4 degrees Celsius with gentle agitation. Use HRP-conjugated anti-rabbit secondary at 1:5000 to 1:10000. Because this is a polyclonal antibody, batch-to-batch titer variation is possible; always titrate with your specific lysate.

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

Triple Point Biologics antibodies are validated for Western blot and many perform well in immunohistochemistry and immunofluorescence, though this specific clone has primary validation for Western blot. For IHC, antigen retrieval is typically required; citrate buffer (pH 6.0) heated retrieval is a reasonable starting point for IDE. Use the antibody at 1:100 to 1:200 dilution. For immunofluorescence, try 1:50 to 1:100 on fixed, permeabilized cells. Because IDE is predominantly cytosolic, you should see diffuse cytoplasmic staining. Include a no-primary-antibody control to assess nonspecific secondary binding, especially in tissue sections.

Does Insulysin have isoforms or splice variants I need to account for?

The IDE gene encodes a single major isoform of 1019 amino acids, and alternative splicing is not prominently reported in the literature. You should see one band near 118 kDa in most lysates. However, IDE can undergo proteolytic cleavage or form homodimers, and these complexes may occasionally appear on gels if not fully denatured. If you observe multiple bands, confirm that samples were boiled adequately in reducing sample buffer. Some studies report mitochondrial or peroxisomal localization of IDE in addition to cytosolic pools, but these are not distinct isoforms. This polyclonal antibody targets a broad epitope range and should detect all forms.

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

Store the antibody at minus 20 degrees Celsius in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. Glycerol or BSA in the formulation buffer helps maintain stability. Once thawed, an aliquot can be kept at 4 degrees Celsius for up to one month if sodium azide (0.02-0.05 percent) is present as preservative; otherwise, store frozen. Do not store diluted antibody; prepare working dilutions fresh for each experiment. The 100 microgram pack size typically provides enough material for 100-200 Western blots at 1:1000 dilution. Antibody stability is generally several years when stored properly.

What sample preparation considerations are important for detecting Insulysin by Western blot?

Use standard RIPA or NP-40 lysis buffer supplemented with protease inhibitors (PMSF, aprotinin, leupeptin) to preserve IDE integrity; the enzyme itself is a protease and may undergo autolysis if inhibitors are omitted. Insulysin is a soluble cytosolic protein, so mild nonionic detergents are sufficient; harsh detergents are unnecessary. Load 20-40 micrograms total protein per lane for abundant cell lines; increase to 50-80 micrograms for low-expression primary cells. Boil samples in reducing Laemmli buffer for 5 minutes. Because IDE is a metalloprotease, avoid EDTA-containing buffers during lysis if you plan functional assays, but EDTA is fine for Western blot sample prep.