NAI1 Antibody

What is NEIL1 and what cellular functions does it perform?

NEIL1 (nei like DNA glycosylase 1) is a crucial DNA repair enzyme belonging to the FPG protein family that primarily functions in base excision repair (BER) pathways. It specifically recognizes and excises DNA damaged by oxidation or mutagenic agents. In humans, the canonical NEIL1 protein consists of 390 amino acid residues with a molecular mass of approximately 43.7 kDa. The protein maintains dual subcellular localization, being present in both the nucleus (where it performs its primary DNA repair functions) and cytoplasm, and is ubiquitously expressed across numerous tissue types. NEIL1 possesses bifunctional activity, acting as both a DNA glycosylase that removes damaged bases and an AP lyase that cleaves the DNA backbone at abasic sites, creating single-strand breaks that signal subsequent repair mechanisms .

What alternative nomenclature exists for NEIL1 protein?

Researchers should be aware of several synonyms when searching literature about NEIL1:

• DNA endonuclease eight-like glycosylase 1

• DNA glycosylase/AP lyase Neil1

• DNA-(apurinic or apyrimidinic site) lyase Neil1

• NEH1

• Endonuclease VIII

• Endonuclease 8-like 1

These alternative designations may appear in different publications and databases, particularly in earlier research before nomenclature standardization.

How conserved is NEIL1 across species?

NEIL1 demonstrates significant evolutionary conservation, with orthologs identified in numerous vertebrate species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . This conservation underscores the fundamental importance of NEIL1 in DNA repair mechanisms across evolutionary lineages and provides various model organisms for comparative studies of NEIL1 function.

What applications are NEIL1 antibodies most commonly used for?

NEIL1 antibodies are primarily employed in Western blotting (WB) applications for detecting NEIL1 protein expression in cellular and tissue extracts. This application allows researchers to quantify NEIL1 levels and assess protein modifications. Enzyme-linked immunosorbent assay (ELISA) represents another common application, particularly useful for quantitative measurement of NEIL1 in solution . When selecting a NEIL1 antibody, researchers should verify that it has been validated for their specific application of interest, as performance can vary significantly between techniques.

How can researchers validate NEIL1 antibody specificity?

Validation of NEIL1 antibody specificity requires a multi-faceted approach:

• Positive and negative controls: Test the antibody on samples with known NEIL1 expression versus those with NEIL1 knockdown or knockout.

• Cross-reactivity testing: Assess potential cross-reactivity with related DNA glycosylases (OGG1, NEIL2, NEIL3) through Western blot analysis.

• Peptide competition assays: Pre-incubate the antibody with purified NEIL1 peptide before application to confirm binding specificity.

• Immunoprecipitation followed by mass spectrometry: Verify that the antibody captures the intended target.

• Multiple antibody comparison: Use antibodies targeting different epitopes of NEIL1 to confirm consistent results.

This systematic validation is essential because non-specific binding can lead to misinterpretation of experimental results, especially when examining subtle changes in NEIL1 expression.

What factors should guide epitope selection when choosing a NEIL1 antibody?

Epitope selection significantly impacts experimental outcomes. Consider these factors:

• Functional domains: Antibodies targeting the N-terminal region may detect NEIL1 catalytic activity, while C-terminal directed antibodies might recognize protein-protein interaction domains.

• Post-translational modifications: Some epitopes may be masked by phosphorylation, acetylation, or other modifications.

• Protein conformation: Certain antibodies may preferentially recognize native versus denatured protein.

• Species cross-reactivity: If comparative studies across species are planned, select antibodies recognizing conserved epitopes.

• Isoform specificity: Ensure the antibody recognizes all relevant NEIL1 isoforms or specifically targets isoforms of interest.

For immunohistochemistry applications, antibodies recognizing formalin-resistant epitopes are preferable, while for co-immunoprecipitation studies, antibodies that do not interfere with protein-protein interactions should be selected.

What are the optimal conditions for Western blot detection of NEIL1?

Successful Western blot detection of NEIL1 requires careful optimization:

Pre-clearing lysates and using fresh protease inhibitors are critical due to potential degradation of NEIL1. Additionally, when analyzing DNA damage response, consider that NEIL1 levels may fluctuate depending on cell cycle phase and stress conditions, necessitating appropriate experimental timing and controls.

How can researchers effectively study NEIL1 interactions with damaged DNA?

Studying NEIL1-DNA interactions requires specialized approaches:

• Electrophoretic mobility shift assays (EMSA): Use synthetic oligonucleotides containing specific lesions (8-oxoguanine, thymine glycol) to assess binding affinity.

• DNA glycosylase activity assays: Measure NEIL1 enzymatic activity using fluorescently labeled substrates containing oxidative lesions.

• Chromatin immunoprecipitation (ChIP): Employ NEIL1 antibodies to identify genomic regions where NEIL1 binds in vivo, particularly after oxidative stress.

• Fluorescence microscopy: Use immunofluorescence with NEIL1 antibodies to track recruitment to DNA damage sites in real-time.

• BER reconstitution assays: Reconstitute the BER pathway in vitro with purified components to study NEIL1's role in the complete repair process.

How can NEIL1 antibodies be used to investigate the relationship between NEIL1 and cancer?

NEIL1 antibodies enable multifaceted investigation of NEIL1's role in cancer:

• Expression analysis: Compare NEIL1 levels in tumor versus normal tissues using immunohistochemistry and Western blotting to identify potential biomarker applications.

• Mutation impact assessment: Study how cancer-associated NEIL1 variants affect protein stability and localization using mutation-specific antibodies.

• DNA repair kinetics: Analyze NEIL1 recruitment to damaged DNA in cancer cells with defective repair pathways.

• Therapeutic response prediction: Correlate NEIL1 expression with sensitivity to radiation or chemotherapy that induces oxidative DNA damage.

• Post-translational modification analysis: Investigate cancer-specific modifications of NEIL1 using modification-specific antibodies.

These approaches are particularly relevant as NEIL1 dysfunction has been implicated in colorectal, gastric, and other cancers, potentially contributing to genomic instability and mutagenesis.

What techniques can be used to study NEIL1 post-translational modifications?

Post-translational modifications (PTMs) critically regulate NEIL1 function. Their study requires specialized techniques:

For comprehensive PTM profiling, immunoprecipitate NEIL1 using validated antibodies followed by mass spectrometry analysis. When investigating stimulus-dependent modifications, include appropriate time course experiments and pathway inhibitors to establish regulatory mechanisms. The challenge lies in distinguishing physiologically relevant modifications from artifacts, requiring careful experimental design and controls.

How can NEIL1 antibodies be used in studying the role of NEIL1 in neurodegenerative diseases?

NEIL1 has emerging roles in neurodegenerative disorders, where oxidative DNA damage accumulation is a pathological feature. NEIL1 antibodies facilitate:

• Comparative expression analysis: Examine NEIL1 levels in affected versus healthy brain regions using immunohistochemistry on post-mortem tissues.

• Co-localization studies: Investigate NEIL1 association with disease-specific protein aggregates (Aβ, tau, α-synuclein) through confocal microscopy and proximity ligation assays.

• Oxidative stress response: Monitor NEIL1 recruitment to damaged DNA in neuronal models under oxidative stress conditions.

• Animal model validation: Track NEIL1 expression changes in rodent models of neurodegeneration using tissue-specific immunoassays.

• Human biomarker development: Assess if NEIL1 levels or post-translational modifications in cerebrospinal fluid correlate with disease progression.

These approaches can provide insights into whether NEIL1 dysfunction contributes to neurodegeneration or represents a compensatory response to increased oxidative damage.

What are common issues when working with NEIL1 antibodies and how can they be resolved?

Researchers frequently encounter these challenges when working with NEIL1 antibodies:

• Weak or absent signal:

  • Increase antibody concentration or incubation time

  • Use alternative extraction methods to improve NEIL1 solubilization

  • Add protease inhibitors to prevent degradation

  • Consider signal amplification techniques

• Multiple bands or non-specific binding:

  • Increase washing stringency and duration

  • Optimize blocking conditions (try different blocking agents)

  • Pre-adsorb antibody with cell lysates from NEIL1 knockout cells

  • Test antibodies targeting different epitopes

• Inconsistent results between experiments:

  • Standardize lysate preparation methods

  • Use internal loading controls

  • Consider that NEIL1 expression fluctuates with cell cycle and stress conditions

  • Maintain consistent experimental timing

• Poor immunoprecipitation efficiency:

  • Optimize antibody-to-bead ratio

  • Adjust salt and detergent concentrations in buffers

  • Consider cross-linking antibody to beads

  • Use gentle elution conditions to preserve protein integrity

Maintaining detailed protocols and systematically testing variables when troubleshooting will help establish reliable NEIL1 detection methods.

How should researchers analyze contradictory data regarding NEIL1 expression or function?

When confronted with contradictory NEIL1 data, implement this analytical framework:

Contradictions in literature may reflect genuine biological complexity rather than experimental error, as NEIL1 regulation and function are highly context-dependent.

What controls are essential when using NEIL1 antibodies in oxidative stress research?

Oxidative stress research with NEIL1 antibodies requires rigorous controls:

• Positive controls:

  • Cells treated with known inducers of oxidative stress (H₂O₂, paraquat)

  • Recombinant NEIL1 protein as Western blot standard

  • Cell lines with verified high NEIL1 expression

• Negative controls:

  • NEIL1 knockout or knockdown cells/tissues

  • Secondary antibody-only controls

  • Peptide competition assays

• Experimental controls:

  • Time course analysis to capture dynamic NEIL1 responses

  • Dose-dependent oxidative stress application

  • Antioxidant co-treatment to confirm specificity

  • Parallel analysis of other DNA glycosylases (OGG1, NEIL2) to differentiate specific versus general BER responses

• Quantification controls:

  • Multiple housekeeping proteins as loading references

  • Standard curves for quantitative applications

  • Normalization to total cell number for cell-specific comparisons

These controls help distinguish genuine NEIL1 responses from experimental artifacts and provide context for interpreting oxidative stress-induced changes in NEIL1 expression or activity.

How might NEIL1 antibodies contribute to understanding the role of NEIL1 in mitochondrial DNA repair?

NEIL1 antibodies are instrumental in elucidating NEIL1's emerging role in mitochondrial DNA maintenance:

• Subcellular fractionation studies: Using NEIL1 antibodies to quantify the proportion of NEIL1 in mitochondrial versus nuclear fractions under various cellular conditions.

• Super-resolution microscopy: Employing fluorescently-labeled NEIL1 antibodies alongside mitochondrial markers to visualize NEIL1 recruitment to mitochondria during oxidative stress.

• Mitochondrial ChIP-seq: Adapting chromatin immunoprecipitation techniques with NEIL1 antibodies to identify binding sites within mitochondrial DNA.

• Proximity labeling approaches: Combining NEIL1 antibodies with techniques like BioID or APEX2 to identify mitochondria-specific NEIL1 interacting partners.

• Post-translational modification profiling: Investigating whether mitochondrial NEIL1 undergoes distinct modifications compared to its nuclear counterpart.

This research direction is particularly significant as mitochondrial DNA is continuously exposed to reactive oxygen species generated during oxidative phosphorylation, making efficient repair mechanisms critical for cellular homeostasis.

What role can NEIL1 antibodies play in investigating NEIL1's potential functions beyond DNA repair?

NEIL1 antibodies are enabling exploration of non-canonical NEIL1 functions:

• RNA metabolism: Investigating potential NEIL1 interactions with RNA processing factors using co-immunoprecipitation and mass spectrometry.

• Transcriptional regulation: Employing ChIP-seq with NEIL1 antibodies to identify potential regulatory roles at gene promoters independent of DNA damage.

• Protein-protein interaction networks: Using NEIL1 antibodies in proximity ligation assays to map NEIL1's interactome under different cellular conditions.

• Stress granule association: Examining NEIL1 localization during cellular stress using immunofluorescence co-localization with stress granule markers.

• Inflammatory signaling: Investigating potential NEIL1 involvement in cytoplasmic DNA sensing and innate immune responses through co-immunoprecipitation with relevant signaling components.

These emerging research directions suggest NEIL1 may serve as a multifunctional protein beyond its established DNA repair activity, potentially connecting genome maintenance with broader cellular processes.