Anti-Elastase-2 Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Neutrophil elastase (ELANE) migrates at approximately 29-30 kDa on reducing SDS-PAGE, corresponding to the mature enzyme after removal of the 19-amino acid signal peptide. The full-length precursor (preproelastase) is 267 amino acids, but the active form released from azurophilic granules is the processed protease. Some researchers observe a slightly higher apparent molecular weight (32-33 kDa) depending on gel percentage and glycosylation status. If you see bands above 50 kDa, consider oligomerization or non-specific binding; include appropriate reducing agents and boil samples thoroughly.

Does this antibody cross-react with other elastase family members or serine proteases?

Human neutrophil elastase shares structural homology with proteinase-3 and cathepsin G, which are also stored in neutrophil granules. While this antibody is validated for human ELANE, cross-reactivity with closely related elastase family members has not been systematically ruled out. If you work with samples containing mixed serine proteases—such as whole neutrophil lysates or inflammatory exudates—run parallel blots with recombinant proteinase-3 and cathepsin G to assess specificity. Predicted cross-reactivity with mouse or rat elastase remains unvalidated; confirmation experiments are advisable if working with rodent samples.

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

Start at 1:1000 dilution in blocking buffer (5% non-fat milk or BSA in TBST) for Western blot analysis of human neutrophil lysates or recombinant ELANE. This polyclonal antibody typically yields clean bands at this dilution with standard chemiluminescent detection. If signal is weak in samples with low elastase expression—such as non-activated neutrophils or certain cell lines—titrate up to 1:500. For plasma or serum samples where elastase is secreted at lower concentrations, consider concentrating your sample or using 1:500 as your starting point and optimize from there.

Can I use this antibody to detect elastase in neutrophil extracellular traps by immunofluorescence?

this antibody is validated for Western blot; immunofluorescence validation is in progress applications and suitable for imaging elastase in NETs. After inducing NET formation with PMA or other stimuli, fix cells with 4% paraformaldehyde and permeabilize with 0.1-0.5% Triton X-100. Elastase redistributes from granules to the nucleus during NET formation and co-localizes with extracellular DNA in released traps. Use a starting dilution of 1:100 to 1:200 for IF and co-stain with DAPI or SYTOX to visualize DNA. Include non-stimulated neutrophils as negative controls to distinguish granular versus nuclear-extracellular localization patterns.

What positive and negative control samples work best for validating elastase detection?

Human neutrophils isolated from peripheral blood and activated with PMA, fMLP, or LPS serve as ideal positive controls; elastase is highly abundant in azurophilic granules and readily detectable. HL-60 cells differentiated with DMTA or retinoic acid also express elastase and provide a reproducible cell line model. For negative controls, use cell types lacking myeloid lineage markers—such as HeLa, HEK293, or primary fibroblasts—which do not express neutrophil elastase. Recombinant human ELANE (if available) provides a defined positive control at known concentrations to confirm antibody specificity and estimate endogenous expression levels.

Should I add protease inhibitors when preparing lysates for elastase Western blots?

Yes, include serine protease inhibitors in your lysis buffer. Neutrophil elastase is catalytically active and can degrade both itself and other proteins during sample preparation. Add PMSF (1 mM), aprotinin, or a complete protease inhibitor cocktail immediately before lysis. Work quickly on ice and avoid multiple freeze-thaw cycles, which promote elastase autodegradation. Some researchers also include EDTA to chelate calcium and reduce activity of metalloproteases that may indirectly affect elastase stability. Boil samples immediately after adding Laemmli buffer to irreversibly denature the protease and preserve band integrity during storage.

How should I store this polyclonal elastase antibody to maintain long-term stability?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which reduce antibody titre and increase aggregation. Upon receipt, spin briefly to collect contents, then aliquot into volumes matching your typical experiment size (10-50 µL). Addition of 50% glycerol allows storage at -20°C without freezing and permits direct pipetting without thawing. For short-term use (up to one month), diluted working stocks can be stored at 4°C with 0.02-0.05% sodium azide as preservative. Triple Point Biologics has produced proteinase antibodies since 1994; proper storage ensures performance throughout the shelf life.

Does elastase show different expression or localization patterns between resting and activated neutrophils?

In resting neutrophils, elastase resides predominantly in azurophilic (primary) granules with characteristic perinuclear localization by immunofluorescence. Upon activation with inflammatory stimuli, elastase undergoes rapid degranulation—either released extracellularly or translocated to the nucleus. Nuclear translocation is essential for histone cleavage and chromatin decondensation during NET formation. By Western blot, total elastase levels remain relatively constant, but subcellular fractionation reveals redistribution from cytoplasmic granules to nuclear and extracellular compartments. If probing activation status, fractionate samples or use imaging to capture localization changes that blotting alone cannot distinguish.