Anti-Proteinase-3 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Proteinase-3 on a Western blot?

Proteinase-3 runs at approximately 29 kDa on Western blot, corresponding to the mature processed form. The full-length protein is 256 amino acids; however, PRTN3 is synthesized as a preproenzyme and undergoes N-terminal signal peptide cleavage and dipeptide removal during granule targeting in neutrophils. You may occasionally observe higher molecular weight bands around 32-34 kDa representing incompletely processed or glycosylated forms, particularly in myeloid cell lines or immature neutrophil populations. Use purified human neutrophils as a positive control to confirm the expected 29 kDa band.

Does this Proteinase-3 antibody cross-react with mouse or rat samples?

This antibody is validated for human Proteinase-3 detection. Cross-reactivity with mouse or rat PRTN3 has not been experimentally confirmed. Human and mouse PRTN3 share approximately 68 percent sequence identity, with divergence concentrated in surface loops away from the catalytic triad. If you plan to use mouse or rat samples, we recommend running a pilot Western blot at 1:500 and 1:1000 dilutions alongside human neutrophil lysate as a positive control. Lack of signal or non-specific banding would indicate the epitope is not conserved across species.

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

Start at 1:1000 dilution for Western blot applications using standard neutrophil or myeloid cell lysates. This rabbit polyclonal is supplied at 1 mg/mL in 100 µg format. For samples with low PRTN3 expression, such as endothelial cells exposing surface-bound proteinase or non-myeloid cells, you may need to increase antibody concentration to 1:500. Incubate overnight at 4°C in blocking buffer containing 5 percent non-fat milk or BSA. Titre the antibody against your specific sample type, as granule content varies with neutrophil activation state and cell maturity.

Can this antibody detect both granule-stored and membrane-bound Proteinase-3?

Yes, this polyclonal antibody recognizes Proteinase-3 regardless of subcellular localization, including the cytoplasmic granule pool and the fraction expressed on the neutrophil plasma membrane. Membrane-bound PRTN3 associates non-covalently with CD177, a GPI-anchored glycoprotein, and constitutes roughly 10-20 percent of total cellular PRTN3 in resting neutrophils. For immunofluorescence studies distinguishing granule versus surface pools, perform staining on non-permeabilized cells first to label surface PRTN3, then permeabilize and counterstain to visualize the granule compartment. This approach clarifies whether experimental treatments mobilize PRTN3 to the cell surface.

What positive and negative controls should I use for Proteinase-3 experiments?

Use freshly isolated human neutrophils or the promyelocytic cell line HL-60 as positive controls; both express high levels of PRTN3 in azurophilic granules. Differentiated HL-60 cells treated with DMSO or retinoic acid further upregulate proteinase expression. For negative controls, use lymphocytes, fibroblasts, or HEK293 cells, which do not express detectable endogenous PRTN3. If studying ANCA-associated vasculitis samples, include serum from healthy donors alongside patient serum to confirm specificity. Always run a no-primary-antibody control to assess non-specific binding of your secondary reagent.

How should I prepare neutrophil lysates to preserve Proteinase-3 detection?

Lyse neutrophils immediately after isolation in RIPA or similar buffer supplemented with protease inhibitors, but note that PRTN3 itself is a serine protease resistant to many inhibitors. Include 1 mM PMSF or aprotinin to limit autoproteolysis and degradation of other sample proteins. Avoid repeated freeze-thaw cycles, which can cause PRTN3 to aggregate or degrade; aliquot lysates and store at -80°C. If studying secreted or surface PRTN3, collect supernatants or perform surface biotinylation before lysis. Activated neutrophils release PRTN3 into culture medium, so clarify supernatants by centrifugation before analysis.

Does Proteinase-3 have isoforms or splice variants I should be aware of?

The PRTN3 gene produces a single major transcript encoding the 256-residue protein. Unlike neutrophil elastase, which has described splice variants, Proteinase-3 does not have widely recognized isoforms in the literature. However, post-translational modifications do occur: the protein undergoes signal peptide cleavage and removal of an activation dipeptide to generate the mature 29 kDa form. Additionally, some reports describe differential glycosylation states depending on cell type and maturation stage. On Western blot, you should see a single dominant band at 29 kDa unless your sample contains immature myeloid precursors.

How should I store this Proteinase-3 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 titer and increase aggregation. The antibody is supplied in a stabilized buffer at 1 mg/mL; under these conditions, rabbit polyclonal antibodies typically retain activity for at least two years from the date of receipt. For long-term storage beyond two years, keep at -80°C. Once thawed, aliquots can be held at 4°C for up to one month for convenience during active experiments. Do not store diluted antibody in working solutions; prepare fresh dilutions in blocking buffer for each experiment.