ENC1 Antibody

What is ENC1 and what cellular functions does it perform?

ENC1, also known as Kelch-like protein 37 (KLHL37) or Nuclear matrix protein NRP/B, functions as an actin-binding protein involved in the regulation of neuronal process formation and differentiation of neural crest cells. At the molecular level, ENC1 works mechanically as part of the ubiquitin-proteasome pathway by acting as a scaffold protein and possessing E3 ligase activity. It facilitates the ubiquitination and proteasomal degradation of target proteins, thereby regulating protein turnover .

Additionally, ENC1 has been shown to down-regulate transcription factor NF2L2/NRF2 by decreasing the rate of protein synthesis rather than through ubiquitin-mediated proteasomal degradation mechanisms . This multifunctional nature makes ENC1 an important target for research across various physiological and pathological contexts.

How does ENC1 expression relate to cancer biology and what implications does this have for antibody-based detection methods?

Recent comprehensive pan-cancer analysis has revealed that ENC1 plays a protumorigenic role in most cancers, with most cancer tissues exhibiting increased ENC1 expression compared to normal tissues . Research has specifically demonstrated that:

• ENC1 overexpression positively correlates with poor clinical outcomes across multiple cancer types

• Gene Set Enrichment Analysis (GSEA) shows ENC1 is closely associated with tumor-promoting biological functions

• ENC1 exhibits negative correlation with infiltration levels of T cells, activated NK cells, and B cells

• Inhibition of ENC1 expression suppresses proliferation and migration in breast cancer, pancreatic cancer, and glioma cells

For antibody-based detection, these findings suggest that researchers should:

• Consider the tumor microenvironment when interpreting ENC1 staining patterns

• Use appropriate controls to account for differential expression between tumor and adjacent normal tissues

• Consider multiplexed approaches to simultaneously evaluate ENC1 and immune cell markers when studying correlations with the tumor immune microenvironment

What technical challenges exist in detecting ENC1 in pituitary adenomas and how can antibody-based approaches address these issues?

To address these challenges when using anti-ENC1 antibodies:

• Validate antibody sensitivity in low-expressing samples before conducting large-scale studies

• Consider using additional markers to confirm adenoma subtype classification

• Implement quantitative image analysis for objective assessment of immunohistochemical staining

• Use complementary methods (qRT-PCR, Western blot) alongside immunohistochemistry to confirm expression levels

Importantly, research has shown that Ki-67 index above 3% is not significantly associated with tumor invasiveness and ENC1 expression , suggesting that ENC1 provides distinct biological information from traditional proliferation markers.

How can ENC1 antibodies be effectively used in studies investigating the relationship between ENC1 and tumor microenvironment?

Given ENC1's established correlation with tumor microenvironment features, antibody-based approaches require careful methodology:

• Multiplex immunofluorescence protocols:

  • Co-stain ENC1 with markers of specific immune cell populations (CD8+ T cells, NK cells, B cells)

  • Use sequential staining with appropriate controls to prevent cross-reactivity

  • Employ spectral unmixing to resolve overlapping fluorescent signals

• Spatial analysis considerations:

  • Analyze ENC1 expression at the tumor-stroma interface versus tumor core

  • Quantify distances between ENC1-expressing cells and infiltrating immune cells

  • Correlate expression patterns with immunomodulator expression in adjacent sections

• Validation approaches:

  • Confirm antibody specificity using genetic knockdown models

  • Employ multiple antibody clones targeting different ENC1 epitopes

  • Correlate protein detection with mRNA expression using methods like spatial transcriptomics

Since most immunomodulators are positively associated with ENC1 , researchers should consider including key immune checkpoint molecules (PD-1, PD-L1, CTLA-4) in their analysis to establish potential mechanistic relationships.

What are the optimal conditions for using ENC1 antibodies in Western blot applications?

Based on validated protocols, researchers should consider the following parameters for Western blot applications:

For optimal results:

• Include both positive and negative controls

• Consider using reducing conditions

• Verify specificity with blocking peptide competition assays

• Be aware that post-translational modifications may affect migration pattern

How should samples be prepared and processed for immunohistochemical detection of ENC1?

For optimal immunohistochemical detection of ENC1, particularly in neural and cancer tissues, the following methodology is recommended:

• Tissue fixation and processing:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Section at 4-5 μm thickness for optimal antibody penetration

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimize retrieval time (typically 15-20 minutes) for specific antibody clone

• Blocking and antibody incubation:

  • Block with 5% normal serum from the species in which the secondary antibody was raised

  • Use optimal antibody dilution (determined empirically for each lot)

  • Incubate at 4°C overnight for maximum sensitivity

• Controls and validation:

  • Include normal pituitary tissue as a positive control (high ENC1 expression)

  • Use isotype controls to assess background staining

  • Consider dual staining with other markers to evaluate co-expression patterns

This methodology has been successfully employed in studies distinguishing invasive from non-invasive null cell adenomas based on ENC1 expression patterns .

What approaches should be used to validate ENC1 antibody specificity for research applications?

A rigorous validation approach for ENC1 antibodies should include multiple complementary methods:

• Western blot validation:

  • Confirm single band at expected molecular weight (66 kDa)

  • Perform blocking peptide competition assay to verify specificity

  • Test in multiple sample types with known differential expression

• Genetic approaches:

  • Test antibody in ENC1 knockout or knockdown models

  • Compare staining pattern with overexpression systems

  • Verify correlation between protein detection and mRNA levels

• Cross-platform validation:

  • Compare results across multiple detection methods (IHC, IF, WB, ELISA)

  • Use multiple antibodies targeting different epitopes

  • Correlate with functional assays related to ENC1 activity

• Specificity controls:

  • Pre-absorb antibody with recombinant ENC1 protein

  • Test cross-reactivity with related kelch-domain proteins

  • Verify species specificity when working with model organisms

These validation approaches are particularly important given ENC1's role as a potential biomarker in cancer research and its differential expression patterns in specific tumor subtypes .

How can ENC1 antibodies be utilized to investigate the role of ENC1 in cancer progression models?

Based on current research indicating ENC1's protumorigenic role across multiple cancers , antibody-based approaches can be implemented to:

• Characterize expression dynamics during disease progression:

  • Perform temporal analysis of ENC1 expression in cancer development models

  • Correlate expression levels with established markers of disease progression

  • Evaluate subcellular localization changes during malignant transformation

• Functional investigation through immunoprecipitation:

  • Use ENC1 antibodies for co-IP studies to identify interaction partners in cancer cells

  • Analyze post-translational modifications specific to cancer contexts

  • Compare interactome between normal and malignant tissues

• Therapeutic response monitoring:

  • Evaluate changes in ENC1 expression following experimental therapies

  • Use ENC1 as a pharmacodynamic marker in preclinical models

  • Correlate changes in expression with functional readouts of tumor behavior

Given that inhibition of ENC1 expression has been shown to suppress proliferation and migration in multiple cancer cell types , antibody-based detection of ENC1 provides a valuable tool for monitoring therapeutic efficacy in experimental models.

What considerations are important when using ENC1 antibodies in comparison to other neurological biomarkers?

When using ENC1 antibodies alongside other neurological biomarkers, researchers should consider:

• Expression pattern overlaps:

  • ENC1 functions in neuronal process formation, potentially overlapping with other neural development markers

  • Differentiate between ENC1 signals and other neural crest markers through co-localization studies

  • Consider temporal expression patterns during development when designing experimental timelines

• Technical compatibility:

  • Ensure antibody host species compatibility for multiplex applications

  • Optimize antigen retrieval conditions that work for all target proteins

  • Select fluorophores with minimal spectral overlap for co-localization studies

• Interpretation challenges:

  • ENC1 has differential expression in normal versus pathological contexts

  • Consider baseline expression levels when interpreting pathological changes

  • Account for regional variations in expression within neural tissues

Unlike the autoantibody testing for encephalopathy (which also uses the acronym ENC1 as a test ID) , research on the ENC1 protein focuses on its intrinsic expression rather than autoimmune responses against neural antigens, requiring distinct experimental approaches and interpretation frameworks.

What are common pitfalls in ENC1 antibody-based experiments and how can they be addressed?

Researchers working with ENC1 antibodies should be aware of these common challenges:

ENC1 expression varies significantly between normal and malignant tissues, and between different adenoma subtypes , necessitating careful optimization and controls for each experimental context.

What quality control measures should be implemented when working with ENC1 antibodies across different experimental platforms?

To ensure reliable and reproducible results when working with ENC1 antibodies:

• Antibody characterization:

  • Document lot-to-lot variation through standardized testing

  • Maintain records of validation experiments for each new lot

  • Consider monoclonal antibodies for applications requiring high reproducibility

• Sample preparation standardization:

  • Develop consistent protocols for tissue processing and storage

  • Document fixation times and conditions for each sample

  • Use automated systems where possible to reduce technical variability

• Quantification and analysis:

  • Implement digital image analysis with defined thresholds for positivity

  • Use reference standards across experimental batches

  • Document software settings and analysis parameters

• Reporting standards:

  • Follow minimum information guidelines for antibody-based experiments

  • Document all experimental conditions, including antibody catalog numbers and dilutions

  • Include representative images of positive and negative controls

These quality control measures are particularly important when using ENC1 as a potential biomarker for distinguishing invasive from non-invasive tumors or when investigating its role in cancer progression .