Anti-Elastase-3A Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for CELA3A on a Western blot?

CELA3A migrates at approximately 28–30 kDa on reducing SDS-PAGE, corresponding to the mature enzyme after cleavage of the signal peptide and activation peptide. The full-length preproenzyme is synthesized at roughly 32 kDa but is processed in the secretory pathway. In pancreatic lysates or conditioned media from pancreatic acinar cells, you may observe both the zymogen and mature forms depending on activation state. Commercial recombinant CELA3A is typically supplied as the mature form. Always run a positive control lysate to confirm the expected migration pattern in your system.

Does this antibody cross-react with other elastase family members like CELA3B or CELA2A?

CELA3A shares high sequence homology with CELA3B (approximately 92 percent identity) and moderate similarity with CELA2A and other chymotrypsin-like proteases in the elastase cluster on chromosome 1p36. Cross-reactivity with closely related isoforms is therefore possible and has not been exhaustively mapped for this polyclonal reagent. If isoform discrimination is critical, confirm specificity by siRNA knockdown of CELA3A or by comparing signal in cell lines known to express only one family member. Validated reactivity is confirmed for human CELA3A; cross-reactivity with mouse or rat orthologs is predicted but not experimentally verified.

What is the recommended starting dilution for Western blot with this Elastase-3A antibody?

The validated dilution for Western blot is 1:1000 in blocking buffer, typically five percent nonfat milk or BSA in TBST. This polyclonal was raised in rabbit, so use an HRP-conjugated anti-rabbit IgG secondary at the manufacturer's recommended dilution. Because CELA3A is expressed at high levels in pancreatic tissue but may be low-abundance in other contexts, you may need to optimize between 1:500 and 1:2000 depending on sample source and protein loading. Incubate primary antibody overnight at four degrees Celsius for maximal sensitivity.

Which tissues or cell lines are good positive controls for CELA3A expression?

Pancreatic tissue or pancreatic acinar cell lysates are the most robust positive controls, as CELA3A is synthesized primarily in the exocrine pancreas and secreted into the duodenum as part of the digestive enzyme repertoire. Human pancreatic carcinoma lines such as PANC-1 or MIA PaCa-2 often retain some CELA3A expression, though levels vary. Duodenal or small intestinal samples may contain CELA3A due to secretion from the pancreas. Negative controls include tissues with negligible pancreatic enzyme expression, such as brain, skeletal muscle, or lymphoid organs.

Can I use this antibody for immunohistochemistry on pancreatic tissue sections?

Yes, Triple Point Biologics antibodies are validated for immunohistochemistry. For CELA3A detection in formalin-fixed paraffin-embedded pancreatic sections, antigen retrieval is typically required; citrate buffer pH 6.0 with heat-induced epitope retrieval is a standard starting point. CELA3A localizes to pancreatic acinar cells, where it is synthesized and stored in zymogen granules. Expect cytoplasmic and granular staining in acinar compartments. Optimize antibody dilution empirically, starting around 1:200 to 1:500. Include a pancreatic tissue section with known CELA3A expression as a positive control.

Does CELA3A have elastolytic activity, and will substrate specificity affect my experiments?

Despite the elastase nomenclature, CELA3A exhibits limited elastolytic activity compared to neutrophil elastase or pancreatic elastase-1. CELA3A functions primarily as an efficient protease with marked specificity for peptide bonds adjacent to alanine residues, not elastin. If your experimental goal involves measuring elastin degradation, CELA3A is not the appropriate target. However, CELA3A is relevant for studies of pancreatic enzyme biology, digestive protease cascades, and substrate specificity within the chymotrypsin fold. Recombinant CELA3A or pancreatic lysates can serve as functional controls if you are assaying proteolytic activity.

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

Store the antibody at minus twenty degrees Celsius in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. The 100 microgram pack size is suitable for dividing into ten to twenty aliquots depending on typical usage. Lyophilized or glycerol-stabilized formulations may tolerate short-term storage at four degrees Celsius; consult the product datasheet. Under proper storage conditions, polyclonal antibodies from Triple Point Biologics typically retain activity for at least two years. Always centrifuge thawed aliquots briefly before use to pellet any aggregates that may have formed.

What sample preparation steps are critical for detecting CELA3A in pancreatic lysates?

Pancreatic tissue is rich in digestive proteases that can degrade target proteins during lysis. Work quickly on ice and include a broad-spectrum protease inhibitor cocktail with serine protease inhibitors such as PMSF or aprotinin. CELA3A itself is a serine protease, so inhibitors will not affect the epitope but will protect other proteins in the lysate. Use RIPA or similar denaturing lysis buffer to ensure complete solubilization. If preparing lysates from frozen tissue, pulverize under liquid nitrogen before adding lysis buffer. Load 20 to 50 micrograms total protein per lane; CELA3A is abundant in pancreas, so signal should be robust.