Anti-Azurocidin Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Azurocidin (AZU1/CAP37) migrates at approximately 29-37 kDa on reducing SDS-PAGE, depending on glycosylation status. The predicted molecular weight from the 251-amino acid sequence is roughly 27 kDa, but azurocidin is a glycoprotein and post-translational modifications shift the apparent size upward. In neutrophil lysates, you may observe a predominant band around 29-32 kDa. Variability between cell types or activation states can alter glycosylation patterns, so a slightly broader or diffuse band is not uncommon. Always include a positive control lysate from neutrophils or differentiated myeloid cells to confirm expected migration.

Does this azurocidin antibody cross-react with other azurophil granule proteins like neutrophil elastase or proteinase 3?

Azurocidin shares structural homology with neutrophil elastase, proteinase 3, and cathepsin G—all members of the azurophil granule serine protease family—but it lacks catalytic activity. This antibody is raised against azurocidin-specific epitopes and has been validated in human neutrophil lysates where all four proteins are co-expressed. We have not observed cross-reactivity with elastase or proteinase 3 in Western blot at the recommended 1:1000 dilution. If you work with crude granule preparations or high-concentration samples, run parallel blots with proteinase 3 or elastase antibodies to confirm specificity in your experimental system.

What starting dilution should I use for Western blot with neutrophil lysates?

Start at 1:1000 dilution in 5% non-fat milk or BSA in TBST for neutrophil or myeloid cell lysates. Azurocidin is abundant in neutrophil azurophil granules, so signal intensity is typically strong at this dilution. If you are working with serum, plasma, or conditioned media where secreted azurocidin (heparin-binding protein) is present at lower concentrations, begin at 1:500 and titrate upward to 1:2000 based on background. Incubate primary antibody overnight at 4°C for optimal sensitivity. Non-myeloid tissues or cell lines may require concentration or enrichment steps before detection.

Will this antibody detect azurocidin in mouse or rat samples?

This antibody has been validated for human azurocidin. Cross-reactivity with mouse or rat orthologs is predicted based on sequence homology—human AZU1 shares approximately 60-65% identity with murine azurocidin—but we have not formally validated reactivity in rodent samples. If you are working with mouse or rat neutrophils, test the antibody at 1:500 to 1:1000 and include a human neutrophil lysate as a positive control on the same blot. Expect weaker signal or potential lack of recognition due to divergence in epitope regions. Consider a species-specific antibody if cross-reactivity is not confirmed in your hands.

What positive and negative controls should I include when detecting azurocidin?

Use human neutrophil lysates or differentiated HL-60 cells as positive controls—azurocidin is highly expressed in azurophil granules and should produce a clear 29-37 kDa band. For a negative control, use cell lines that lack myeloid lineage markers, such as HeLa, HEK293, or Jurkat T cells, which do not express azurocidin under standard culture conditions. If you are studying azurocidin release or secretion, include unstimulated and stimulated neutrophil supernatants to demonstrate degranulation-dependent detection. Recombinant azurocidin is also a useful positive control if you need to confirm antibody specificity independent of cellular context.

Can I use this antibody for immunohistochemistry on bone marrow or inflamed tissue?

Yes, this antibody is validated for Western blot; IHC validation is in progress and suitable for paraffin-embedded or frozen sections of human bone marrow, inflamed tissue, or infection sites where neutrophil infiltration occurs. Azurocidin localizes to neutrophil cytoplasmic granules, so you should observe punctate cytoplasmic staining in mature neutrophils and myeloid precursors. Antigen retrieval is recommended for paraffin sections—citrate buffer (pH 6.0) at sub-boiling temperature for 20 minutes is a standard starting point. Titrate antibody dilution beginning at 1:100 to 1:200. Expect positive staining in granule-rich myeloid cells and possible extracellular staining in areas of active neutrophil degranulation.

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

Azurocidin is encoded by a single-copy gene (AZU1) on chromosome 19 and does not have well-characterized splice variants or isoforms that would alter protein size significantly. The primary variation you may encounter is in glycosylation, which can cause band shifts of 2-5 kDa depending on neutrophil activation state or tissue source. Some studies report proteolytic processing or complexing with heparin or other glycosaminoglycans in extracellular environments, which could alter migration or detection. In standard lysates, expect a single predominant band. If you observe multiple bands, consider deglycosylation with PNGase F to confirm glycosylation heterogeneity versus degradation or non-specific binding.

How should I store this antibody and what is the expected shelf life?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. The antibody is supplied in a stabilized buffer at 100 µg per vial. Once thawed, aliquots can be kept at 4°C for up to one month if used frequently, but long-term storage should remain frozen. Expect shelf life of at least 12 months from date of receipt when stored properly at -20°C. Do not store diluted antibody in working solutions for more than one week. If you observe increased background or loss of signal over time, prepare a fresh aliquot and re-titre against your positive control lysate.