CELA3A Antibody

What is CELA3A and what is its biological function?

CELA3A is a pancreatic serine proteinase that digests dietary proteins. Despite its name suggesting elastolytic properties, it demonstrates only minimal elastolytic activity while showing efficient protease activity with alanine specificity . CELA3A belongs to the chymotrypsin-like elastase family and is also known by several alternative names including Elastase IIIA, Elastase-3A, and Protease E . It functions primarily in the digestive system as a pancreatic enzyme secreted into the small intestine.

How does CELA3A differ from other elastase family members?

CELA3A represents one member of the elastase family with distinct evolutionary characteristics. Unlike some species that express CELA1, the human pancreas does not express CELA1 but instead secretes two CELA3 isoforms - CELA3A and CELA3B . This evolutionary development appears unique to humans, where both isoforms are expressed at comparable levels at both mRNA and protein levels . This duplication may represent a compensatory mechanism for the loss of CELA1 in humans, potentially through increased gene dosage rather than through development of divergent substrate specificities .

What applications are CELA3A antibodies validated for?

CELA3A antibodies have been validated for multiple research applications. For example, rabbit polyclonal CELA3A antibodies such as ab232708 have been validated for Western blot (WB) and immunohistochemistry on paraffin-embedded sections (IHC-P) with confirmed reactivity against human, mouse, and cow samples . Other antibodies like ab21590 have additional validations for immunocytochemistry (ICC), immunoprecipitation (IP), and ELISA techniques specifically with mouse samples . Researchers should select antibodies based on their specific experimental needs and the validated applications.

What is known about the substrate specificity of CELA3A compared to CELA3B?

How can researchers experimentally determine CELA3A substrate specificity?

To characterize CELA3A substrate specificity, researchers should consider a multi-method approach:

• Phage display selection: Libraries displaying substrate-like proteinase inhibitors (such as SGPI-2) can be used to select high-affinity binders through multiple rounds of panning .

• Recombinant inhibitor analysis: Based on phage-selected sequence patterns, researchers can produce recombinant SGPI-2 variants with systematically altered residues at key positions (P4, P1, P1', P2', P4') .

• Binding affinity measurements: Equilibrium binding assays can determine dissociation constants (KD) for each variant, providing quantitative measures of binding preference .

• Comparative analysis: Testing the same inhibitor variants against related proteases (CELA3B, CELA1) helps differentiate specific binding characteristics of CELA3A .

This comprehensive approach has successfully revealed that the primary specificity of CELA3A, CELA3B, and porcine CELA1 is similar but with notable differences in extended substrate recognition .

What methodological approaches can differentiate between CELA3A and CELA3B in research samples?

Differentiating between the highly similar CELA3A and CELA3B proteins requires specialized approaches:

• Isoform-specific antibodies: When available, validated antibodies targeting divergent epitopes can distinguish between the isoforms in immunoassays.

• Substrate specificity profiling: Exploiting the subtle differences in substrate preferences, particularly at the P1 position where CELA3A prefers Ile and excludes Met, while CELA3B prefers Leu and tolerates Met .

• Inhibitor-based discrimination: Using the SGPI-2 inhibitor variants with established differential binding affinities can help distinguish CELA3A from CELA3B activity in complex samples .

• Mass spectrometry: Targeted proteomic approaches can identify isoform-specific peptides, particularly from regions with sequence divergence.

What are the optimal conditions for using CELA3A antibodies in Western blot applications?

For successful Western blot detection of CELA3A:

• Sample preparation:

  • Use pancreatic tissue lysates where CELA3A is highly expressed

  • Include protease inhibitors to prevent degradation

  • Load 20-50 μg of total protein per lane

• Electrophoresis and transfer:

  • Use 12-15% gels (CELA3A has predicted band size of 29 kDa)

  • Ensure complete transfer to PVDF or nitrocellulose membranes

• Antibody incubation:

  • Block with 5% non-fat milk or BSA

  • Incubate with primary antibody at 2 μg/mL concentration

  • Use appropriate HRP-conjugated secondary antibody

• Controls:

  • Include recombinant human CELA3A protein as positive control

  • Use pancreatic tissue lysates from relevant species (human, mouse, or cow)

The Western blot should show a single, specific band at approximately 29 kDa, as validated in cow and mouse pancreas lysates with ab232708 .

What protocol is recommended for immunohistochemical detection of CELA3A?

For optimal immunohistochemical visualization of CELA3A in tissue sections:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded tissue sections (FFPE)

  • Human pancreas tissue is recommended as positive control

• Antigen retrieval:

  • Perform appropriate antigen retrieval method (buffer and conditions may need optimization)

  • Allow slides to cool before antibody application

• Primary antibody incubation:

  • Apply CELA3A antibody at appropriate concentration (20 μg/ml recommended for ab232708)

  • Incubate according to manufacturer's protocol

• Detection:

  • Use compatible secondary antibody system

  • Develop with DAB for chromogenic detection

  • Counterstain with hematoxylin for nuclear visualization

Published results show specific staining of CELA3A in pancreatic acinar cells, which should be used as reference for successful staining patterns .

How can researchers verify the specificity of CELA3A antibodies?

Validating antibody specificity is crucial for reliable results, especially given the similarity between CELA3A and CELA3B:

• Recombinant protein controls:

  • Test antibody against purified recombinant CELA3A protein

  • Assess potential cross-reactivity with recombinant CELA3B

• Tissue expression patterns:

  • Confirm expected staining in pancreatic tissue (known to express CELA3A)

  • Use appropriate negative control tissues

• Western blot analysis:

  • Verify single band at expected molecular weight (29 kDa)

  • Compare with predicted band patterns if multiple splice variants exist

• Blocking peptide competition:

  • Pre-incubate antibody with immunizing peptide

  • Specific signal should be abolished or significantly reduced

What are common challenges when working with CELA3A antibodies and how can they be addressed?

How should researchers interpret evolutionary differences when studying CELA3A across species?

When conducting cross-species studies involving CELA3A, researchers should consider several important factors:

• Evolutionary divergence: Humans uniquely express CELA3A and CELA3B but not CELA1, while other species like pigs express CELA1 . This fundamental difference requires careful interpretation of functional studies.

• Antibody cross-reactivity: Verify that antibodies raised against human CELA3A properly recognize the ortholog in other species. For example, ab232708 has been validated to react with human, mouse, and cow samples .

• Substrate specificity variations: The substrate preferences may differ between species. For instance, porcine CELA1 strongly prefers P1 Met, which is poorly recognized by human CELA3 isoforms .

• Functional compensation: In cross-species comparisons, consider that functional roles may be distributed differently among elastase family members due to evolutionary adaptations .

What considerations are important when analyzing CELA3A binding interactions?

Research into CELA3A binding interactions requires attention to several methodological details:

• Binding site architecture: CELA3A has multiple substrate interaction sites (S4, S2, S1, S1', S2', S4') that collectively determine binding specificity .

• Quantitative assessment: Equilibrium binding assays can determine dissociation constants (KD) with purified components, enabling precise comparison between variants .

• Context effects: Consider that binding interactions observed with purified proteins may differ from those in complex biological samples due to competing interactions.

• Experimental design for comparative studies:

  • Use consistent experimental conditions when comparing CELA3A with CELA3B or CELA1

  • Include appropriate controls for binding specificity

  • Consider multiple binding parameters beyond just affinity (e.g., on/off rates)

• Interpretation of inhibitor studies: When using SGPI-2 variants to probe binding specificity, remember these bind in a substrate-like manner but may not perfectly mimic actual substrate interactions .

How can CELA3A antibodies be utilized in pancreatic disease research?

CELA3A antibodies provide valuable tools for investigating pancreatic pathologies:

• Pancreatitis studies:

  • Analyze altered CELA3A expression/localization in inflammatory conditions

  • Investigate premature enzyme activation within acinar cells

  • Examine CELA3A release into circulation as potential biomarker

• Pancreatic cancer research:

  • Compare CELA3A expression between normal and neoplastic tissues

  • Assess potential diagnostic or prognostic value

  • Study possible roles in tumor microenvironment modulation

• Molecular research approaches:

  • Use CELA3A antibodies for protein-protein interaction studies via co-immunoprecipitation

  • Perform immunohistochemical analysis to determine cellular/subcellular localization

  • Conduct Western blot analysis to quantify expression levels in disease models

What role does CELA3A play in the complex with procarboxypeptidases?

Research has revealed interesting differences in the interactions of CELA3 isoforms with other pancreatic enzymes:

• Binary complex formation: Interestingly, the proenzyme of human CELA3B, but not human CELA3A, forms binary complexes with procarboxypeptidase A1 and A2 .

• Evolutionary significance: A similar binary complex has been isolated from pig pancreas, suggesting conserved functional interactions across species .

• Research methodologies:

  • Co-immunoprecipitation using CELA3A antibodies can identify interaction partners

  • Size exclusion chromatography can detect complex formation

  • Functional assays can determine how complex formation affects enzymatic activity

• Physiological implications: These differential interactions may indicate specialized roles for CELA3A versus CELA3B in digestive processes, despite their similar substrate specificities .

How can CELA3A research contribute to understanding human digestive enzyme evolution?

The study of CELA3A offers unique insights into evolutionary adaptations of digestive enzymes:

• Human-specific adaptations: The loss of CELA1 expression and duplication of CELA3 genes appears to be a human-specific evolutionary event .

• Functional compensation hypothesis: Research suggests that increased gene dosage through CELA3 duplication, rather than specificity divergence, may compensate for the loss of CELA1 digestive activity in humans .

• Research approaches:

  • Comparative genomics across species

  • Substrate specificity studies comparing human CELA3A/B with CELA1 from other species

  • Functional assays testing digestion of various protein substrates

• Evolutionary implications: This research contributes to our understanding of how digestive enzyme repertoires adapt to dietary changes throughout human evolution, potentially reflecting adaptations to changing nutritional environments.