Anti-Elastase-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Elastase-1 on Western blot?

CELA2A, the predominant circulating elastase-1 isoform, migrates at approximately 28–30 kDa as the mature enzyme after signal-peptide cleavage. You may also detect the 269-residue zymogen near 29 kDa in pancreatic or plasma samples, since activation by trypsin is tissue-dependent. CELA1 is closely related and runs at a similar size. If you see higher bands around 55–60 kDa, consider dimer formation under non-reducing conditions or incomplete denaturation. Always include a reducing agent and confirm your sample source, since pancreatic tissue expresses both isoforms while plasma predominantly contains CELA2A.

Does this antibody distinguish between CELA1 and CELA2A isoforms?

Most polyclonal elastase-1 antibodies raised against full-length or large fragments recognize epitopes conserved across the chymotrypsin-like elastase family, so they typically detect both CELA1 and CELA2A. The two proteins share high sequence homology within the catalytic domain. If isoform specificity is critical for your experiment, confirm by running recombinant CELA1 and CELA2A standards in parallel lanes, or consider knockdown controls in cell lines expressing one isoform preferentially. Alternatively, check whether the immunogen sequence overlaps unique N- or C-terminal regions, though this information is not always disclosed for polyclonal sera.

What is the recommended starting dilution for Western blot with this Elastase-1 antibody?

Start at 1:1000 in blocking buffer for Western blot, as indicated in the validation data. This dilution works well for lysates from pancreatic tissue or cell lines expressing moderate to high elastase-1 levels. For plasma samples or low-abundance systems, you may need to concentrate your sample or test 1:500. Always include a positive control such as human pancreatic lysate or recombinant CELA2A. If background is high, increase the dilution to 1:2000 and extend blocking time. Titrate against your specific sample type and detection system, since chemiluminescence sensitivity varies across substrates and cameras.

Which species does this Elastase-1 antibody cross-react with besides human?

This antibody is validated for human Elastase-1. Cross-reactivity with mouse, rat, or other mammalian orthologs is not confirmed and should be considered predicted at best. Elastase-1 sequences show moderate conservation across mammals, but divergence in surface loops can abolish polyclonal recognition. If you plan to work with non-human samples, run a pilot blot with lysate from the target species alongside human pancreatic control. Lack of signal does not always mean the ortholog is absent; it may reflect epitope variability. For rodent studies, consider sourcing an antibody explicitly validated in mouse or rat, particularly for IHC or IF applications.

What sample preparation steps are important for detecting Elastase-1 in plasma versus tissue lysates?

For plasma, CELA2A circulates as an active protease, so add protease inhibitors immediately upon collection and keep samples on ice. EDTA or citrate plasma works well; avoid heparin if downstream assays are sensitive to it. For pancreatic tissue, homogenize in lysis buffer containing serine-protease inhibitors such as AEBSF or PMSF, since elastase autoactivation and cross-activation by trypsin can degrade targets during extraction. Clarify lysates by centrifugation at 12,000–16,000 × g and quantify protein with a compatible assay. Elastase-1 is reasonably stable at –80 °C but loses activity through freeze-thaw cycles, so aliquot samples for single use.

What positive and negative controls should I use when validating Elastase-1 detection?

Human pancreatic tissue lysate or juice is the standard positive control, since both CELA1 and CELA2A are abundantly expressed there. Recombinant CELA2A protein serves as a defined-MW standard. For plasma studies, pooled human plasma typically shows a detectable signal. Negative controls include lysates from tissues with negligible elastase expression, such as liver or skeletal muscle, and elastase-knockout cell lines if available. Peptide-competition blocking with the immunogen peptide (if provided) confirms specificity. For IHC, normal pancreas is positive; spleen or lymph node are usually negative, helping distinguish specific signal from non-specific background in your staining protocol.

How should I store this polyclonal Elastase-1 antibody to maintain activity?

Store the antibody at –20 °C as supplied. For frequent use, aliquot into single-use volumes to avoid repeated freeze-thaw cycles, which can aggregate IgG and reduce titer. A working aliquot may be kept at 4 °C for up to one month if sodium azide or another preservative is present; check the datasheet. Do not store diluted antibody in blocking buffer for more than a few days, as carrier proteins like BSA or milk can promote microbial growth. If you observe increased background or loss of signal over time, thaw a fresh aliquot and re-titrate. Rabbit polyclonal sera remain stable for years under these conditions.

Can I use this antibody for immunohistochemistry or immunofluorescence on pancreatic sections?

Yes, Triple Point Biologics antibodies are validated for Western blot; IHC and immunofluorescence validation is in progress. For pancreatic sections, antigen retrieval is typically required: citrate buffer (pH 6.0) heat-induced epitope retrieval works well for most serine proteases. Block endogenous peroxidase if using HRP detection, and apply the primary antibody at a starting dilution around 1:100 to 1:200 overnight at 4 °C. Elastase-1 localizes to zymogen granules in acinar cells, so expect cytoplasmic punctate staining. Optimize retrieval and dilution on serial sections, and include a no-primary-antibody control to assess non-specific secondary binding.