Anti-Elastase-2A Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight of Elastase-2A on Western blot?

Elastase-2A (CELA2A) migrates at approximately 29-30 kDa on reducing SDS-PAGE, corresponding to the mature secreted form after signal peptide cleavage. The full-length protein is 269 amino acids, but the 18-residue signal sequence is removed during secretion, yielding the circulating enzyme. Depending on sample source and post-translational modifications, you may observe minor shifts. Pancreatic lysates and plasma samples typically show a single prominent band. Start with 1:1000 dilution in 5% non-fat milk or BSA in TBST, and confirm identity with appropriate positive controls such as human pancreatic tissue lysate or recombinant CELA2A.

Does this Elastase-2A antibody cross-react with other elastase family members?

This antibody is validated specifically for human CELA2A. The chymotrypsin-like elastase family includes closely related isoforms such as CELA2B, CELA3A, CELA3B, and neutrophil elastase (ELANE), which share significant sequence homology. We have not systematically tested cross-reactivity against these paralogs. If your experiment requires strict isoform specificity, particularly when working with pancreatic or neutrophil samples where multiple elastases are co-expressed, confirm specificity using siRNA knockdown or CELA2A-null lysates as negative controls. Predicted cross-reactivity with non-human primates based on sequence conservation, but experimental validation is recommended for non-human species.

What is the recommended starting dilution for Elastase-2A Western blot?

We recommend starting at 1:1000 dilution for Western blot applications with this rabbit polyclonal antibody. This dilution has been validated for human samples and typically provides clean signal with low background when blocking with 5% milk or BSA in TBST for one hour at room temperature. Incubate primary antibody overnight at 4°C or two hours at room temperature. Depending on expression level in your sample type, you may optimize between 1:500 and 1:2000. Plasma and pancreatic lysates generally express detectable CELA2A; for low-abundance samples like certain cell lines, start at the higher concentration end of the range and titrate downward.

Which tissues and cell types express Elastase-2A at detectable levels?

CELA2A is primarily expressed and secreted by pancreatic acinar cells, making pancreatic tissue the most reliable positive control. The enzyme circulates in plasma at measurable concentrations, so serum or plasma samples are also appropriate for detection. Expression has been reported in certain epithelial tissues and potentially in endocrine contexts related to glucose metabolism, though levels vary considerably. For Western blot validation, use human pancreatic tissue lysate or recombinant CELA2A protein as positive controls. If working with cell lines, verify CELA2A expression via RT-PCR or RNA-seq data before committing to immunoblotting, as many immortalized lines lack detectable expression.

Can I use this antibody for immunohistochemistry on pancreatic tissue?

Yes, This antibody is validated for Western blot; immunohistochemistry/immunofluorescence validation is in progress applications, and pancreatic tissue is an excellent substrate given CELA2A's high expression in pancreatic acinar cells. For IHC on formalin-fixed paraffin-embedded sections, antigen retrieval is typically required—citrate buffer (pH 6.0) heat-mediated retrieval is a reasonable starting point. Use the antibody at dilutions between 1:100 and 1:500 depending on fixation time and tissue preservation quality. Expect strong cytoplasmic staining in acinar cells with minimal background in islets. Include a no-primary-antibody control and consider blocking endogenous peroxidase activity if using HRP-based detection systems.

How should I prepare plasma samples for Elastase-2A detection by Western blot?

CELA2A circulates as a secreted protease in plasma, so sample preparation is straightforward but requires protease inhibitor precautions. Collect plasma in EDTA or citrate tubes and store at -80°C until use. Before Western blot, dilute plasma 1:10 to 1:50 in sample buffer to avoid overloading protein and ensure adequate separation. Because CELA2A is itself a serine protease, include a broad-spectrum protease inhibitor cocktail during sample handling to prevent degradation of other plasma proteins, though CELA2A itself is relatively stable. Load 10-20 µg total protein per lane. Albumin depletion is generally unnecessary but can improve band clarity if background is high.

What is the recommended storage condition for this Elastase-2A antibody?

Store the antibody at -20°C in single-use aliquots to maintain long-term stability and avoid repeated freeze-thaw cycles, which can compromise antibody performance. The antibody is supplied in a stabilized buffer at 100 µg per vial. Upon receipt, aliquot into working volumes suitable for your experimental frequency—typically 10-20 µL per tube—and return immediately to -20°C. For short-term use within two weeks, diluted antibody can be stored at 4°C in the presence of 0.02% sodium azide, though we recommend preparing fresh working dilutions for optimal results. Do not store diluted antibody in blocking buffer for more than one week.

Does Elastase-2A run as a single band or are there multiple isoforms visible?

In most sample types, CELA2A appears as a single band at approximately 29-30 kDa corresponding to the mature, secreted form of the protease. The gene encodes a single primary isoform without common splice variants. However, you may occasionally observe doublets or minor higher-molecular-weight species due to glycosylation, disulfide bond formation under non-reducing conditions, or protein complexes if samples are not fully denatured. Ensure complete reduction with DTT or beta-mercaptoethanol and boiling for five minutes. If multiple bands persist, consider that CELA2A may form complexes with endogenous protease inhibitors like alpha-1-antitrypsin in plasma samples, which could shift apparent molecular weight.