AAE1 Antibody

What specific cytokeratin types does AE1 antibody recognize?

The AE1 monoclonal antibody is specific for acidic (Type I) family cytokeratins. It recognizes several cytokeratin proteins with molecular weights of 56.5, 54', 50, 50', 48, and 40 kDa, which correspond to cytokeratins 10, 14, 15, 16, and 19 . This specificity profile makes AE1 a valuable tool for detecting acidic cytokeratins in epithelial tissues and tumor samples . The antibody's recognition profile has been thoroughly validated across multiple species including human, mouse, rat, and various other mammals.

How does AE1 antibody differ from AE3 and the combined AE1/AE3 antibodies?

AE1 and AE3 antibodies target distinct complementary families of cytokeratins:

The AE1/AE3 cocktail combines both antibodies to provide comprehensive detection of both acidic and basic cytokeratins, significantly enhancing epithelial cell identification in diagnostic applications. This combined approach is particularly valuable in identifying poorly differentiated carcinomas and determining the epithelial origin of neoplasms .

What is the molecular basis for AE1 antibody's specificity?

AE1 antibody was developed using human epidermal keratins as the immunogen . Its specificity stems from recognition of conserved epitopes present in acidic cytokeratins. The antibody binds to specific sequences within the alpha-helical core domain of these cytokeratins, which explains its cross-reactivity across multiple species while maintaining specificity for the Type I cytokeratin family . This molecular recognition mechanism underlies AE1's reliability as a marker for epithelial differentiation.

What are the validated research applications for AE1 antibody?

AE1 antibody has been validated for multiple research applications:

• Western Blotting (WB): Effective at dilutions of 1:1000-1:3000 for detecting cytokeratins in protein lysates

• Immunohistochemistry (IHC):

  • Paraffin-embedded sections (IHC-P)

  • Frozen sections (IHC-Fr)

• Immunocytochemistry (ICC): Useful for cellular localization studies

• Immunofluorescence: When conjugated with fluorophores (e.g., Alexa Fluor 488), supports multicolor imaging applications

For optimal results in any application, empirical titration is recommended to determine the ideal concentration for specific experimental conditions .

How should researchers optimize AE1 antibody protocols for immunohistochemistry?

Successful immunohistochemistry with AE1 antibody requires careful optimization:

• Antigen Retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is typically recommended for formalin-fixed tissues

• Blocking: Use 5% dried milk powder in wash buffer to minimize non-specific binding

• Antibody Concentration: Begin with dilutions between 1:100-1:500 for IHC applications and adjust based on signal intensity and background

• Detection Systems: Compatible with both chromogenic (e.g., DAB) and fluorescent detection systems

• Controls: Always include appropriate positive controls (epithelial tissues) and negative controls (omitting primary antibody)

Optimization is particularly important when working with archival tissues or samples subjected to extended fixation times, as these conditions may affect epitope accessibility.

What methodological considerations are important for Western blot applications of AE1 antibody?

When using AE1 antibody for Western blotting:

• Sample Preparation: Complete solubilization of intermediate filaments requires strong denaturing conditions; use buffers containing SDS and reducing agents

• Gel Percentage: Use 10-12% polyacrylamide gels to achieve optimal separation of different cytokeratin proteins (40-56.5 kDa)

• Antibody Dilution: Start with 1:1000-1:3000 dilution in blocking buffer containing 5% dried milk powder

• Validation: Test dilutions on established cell lines with known cytokeratin expression (e.g., MCF7 cells)

• Visualization: Compatible with standard chemiluminescent detection systems

For optimal detection of all cytokeratin species, researchers should ensure complete protein transfer to the membrane and may need to adjust exposure times accordingly.

How can AE1 antibody be utilized in cancer research and diagnostics?

AE1 antibody serves as a valuable tool in cancer research and diagnostics:

• Tumor Classification: Helps identify epithelial origin of poorly differentiated tumors by detecting cytokeratin expression patterns

• Metastasis Detection: Identifies epithelial cells in non-epithelial tissues (e.g., lymph nodes) indicating metastatic spread

• Tumor Heterogeneity Studies: Characterizes cytokeratin expression patterns within tumor populations

• Circulating Tumor Cell (CTC) Detection: Aids in identifying epithelial-derived CTCs in blood samples

• Biomarker Development: Can be incorporated into antibody panels for early cancer detection

Researchers have demonstrated that cytokeratin expression patterns detected by AE1 can provide insights into tumor differentiation status and cellular origin, contributing to more accurate cancer classification and potentially guiding treatment decisions .

What is the significance of AE1 antibody in antibody developability studies?

In antibody engineering and development research:

• Model Antibody: AE1 serves as a well-characterized model antibody for studies on antibody structure-function relationships

• Developability Assessment: Provides insights for predicting antibody performance in downstream processes and formulations

• Screening Funnel Development: Contributes to understanding how antibody characteristics affect their progression through screening funnels during therapeutic antibody development

• Manufacturing Considerations: Offers lessons about antibody stability, aggregation propensity, and other critical quality attributes

Antibody developability studies, as demonstrated in high-throughput workflows, evaluate key parameters that predict antibody behavior during manufacturing and storage. AE1's well-documented characteristics make it valuable for comparative studies in this emerging field .

How can AE1 antibody be used in multiplex immunofluorescence panels?

Advanced multiplex immunofluorescence applications using AE1 antibody:

• Panel Design: Consider AE1's mouse IgG1 isotype when designing multiplex panels to avoid cross-reactivity with other primary antibodies

• Fluorophore Selection: AE1 conjugated to Alexa Fluor 488 works effectively; select complementary fluorophores for other markers based on spectral compatibility

• Sequential Staining: For complex panels, utilize sequential staining approaches with appropriate blocking between steps

• Co-localization Studies: Combine AE1 with antibodies against other cellular components to study intracellular organization of cytokeratins

• Tissue Microenvironment Analysis: Use AE1 to identify epithelial cells while simultaneously characterizing surrounding stromal and immune components

When designing multiplex panels, researchers should validate antibody performance in single-color experiments before combining multiple antibodies to ensure specificity and sensitivity are maintained in the multiplex format.

What are common challenges when using AE1 antibody, and how can they be addressed?

Researchers may encounter several challenges when working with AE1 antibody:

• High Background:

  • Increase blocking time/concentration (use 5% milk powder in wash buffer)

  • Verify secondary antibody specificity

  • Optimize antibody dilution (1:1000-1:3000 for WB)

  • Include additional washing steps

• Weak Signal:

  • Improve antigen retrieval methods for fixed tissues

  • Reduce antibody dilution

  • Extend primary antibody incubation time

  • Consider signal amplification systems

• Non-specific Bands in Western Blot:

  • Increase blocking stringency

  • Optimize SDS-PAGE conditions

  • Verify sample preparation methods

  • Consider using gradient gels for better separation

• Inconsistent Results:

  • Standardize fixation protocols

  • Control incubation temperatures

  • Prepare fresh working dilutions

  • Aliquot antibody to avoid freeze-thaw cycles

How should species cross-reactivity be validated when using AE1 antibody in non-human samples?

When extending AE1 antibody use to non-human species:

• Positive Control Selection: Include known positive controls from the target species alongside human samples

• Sequence Homology Analysis: Evaluate cytokeratin sequence conservation between human and target species

• Dilution Optimization: Titrate antibody concentrations specifically for each species

• Alternative Validation Methods: Confirm findings with species-specific antibodies or other detection methods

• Western Blot Verification: Confirm appropriate molecular weight bands in the target species

AE1 antibody has documented reactivity with mouse, rat, rabbit, chicken, cow, pig, and monkey samples , but optimization is still required for each specific application and tissue type across species.

What considerations are important when analyzing conflicting or unexpected results with AE1 antibody?

When encountering unexpected or conflicting results:

• Epitope Accessibility: Consider whether tissue processing or fixation methods may mask target epitopes

• Isoform Expression: Verify which specific cytokeratin isoforms are expected in your sample type

• Disease State Influence: Consider how pathological conditions might alter cytokeratin expression patterns

• Cross-Reactivity: Evaluate potential cross-reactivity with non-target proteins in your specific sample context

• Methodology Comparison: Compare results across different detection methods (IHC, WB, ICC)

• Technical Validation: Reproduce experiments with different lots of antibody or alternative anti-cytokeratin antibodies

Systematic validation using multiple methodological approaches can help resolve conflicting results and determine whether unexpected findings represent biological variation or technical artifacts.

How is AE1 antibody being integrated into emerging cancer biomarker research?

Current research is exploring innovative applications of AE1 antibody in cancer biomarkers:

• Liquid Biopsy Development: AE1 is being employed to detect cytokeratin-positive circulating tumor cells in blood samples

• Biomarker Panels: Studies are incorporating AE1 into multiplexed antibody panels for early cancer detection and monitoring

• Tumor Microenvironment Analysis: Research is utilizing AE1 alongside other markers to characterize epithelial-mesenchymal transitions

• Predictive Biomarkers: Investigations are evaluating whether specific cytokeratin profiles detected by AE1 can predict treatment response

• Minimal Residual Disease Detection: Emerging applications focus on identifying rare epithelial cells in post-treatment samples

As reviewed in recent literature, antibodies against tumor-associated antigens like cytokeratins show significant potential as diagnostic biomarkers, particularly when used in panels rather than as single markers .

What are the technical advances improving AE1 antibody applications in research?

Technological advancements enhancing AE1 antibody utility include:

• Recombinant Antibody Production: Improving consistency and reducing batch-to-batch variation

• Novel Conjugation Chemistries: Enabling site-specific attachment of fluorophores, enzymes, or nanoparticles

• Automated Staining Platforms: Standardizing immunohistochemical applications

• Single-Cell Analysis Integration: Incorporating AE1 into single-cell protein profiling methods

• High-Throughput Screening Applications: Utilizing AE1 in large-scale antibody developability assessments

• Computational Analysis Tools: Improving quantitation and interpretation of cytokeratin expression patterns

These technological advances are transforming how researchers utilize AE1 antibody, enabling more precise, reproducible, and high-dimensional analyses of cytokeratin expression in both basic research and clinical applications.