NEURL2 Antibody

What is NEURL2 and why is it a target for antibody-based detection?

NEURL2 (Neuralized E3 Ubiquitin Protein Ligase 2) functions as a ubiquitin ligase involved in protein degradation pathways. This protein has emerged as an important research target due to its association with neurological processes and potential implications in coronary artery disease genetics, as identified in recent genome-wide association studies . Antibody-based detection of NEURL2 enables researchers to study its expression patterns, protein interactions, and functional roles in various experimental contexts through immunoblotting, immunostaining, and immunoprecipitation approaches.

What applications are NEURL2 antibodies validated for in research?

Current NEURL2 antibodies have been validated for multiple research applications with varying degrees of optimization. The primary applications include:

It is important to note that performance correlation between applications varies significantly; success in IF appears to be the best predictor of performance in WB and IP applications .

How should I select the appropriate NEURL2 antibody for my specific experimental design?

When selecting a NEURL2 antibody, consider these methodological criteria:

• Application compatibility: Verify that the antibody has been specifically validated for your intended application (WB, IHC, IF, etc.). Research indicates that 20-30% of protein studies may use ineffective antibodies , making validation critical.

• Epitope specificity: Different NEURL2 antibodies target specific regions:

  • N-terminal region antibodies (e.g., AA 99-148)

  • Central region antibodies

  • Internal region antibodies

  • C-terminal region antibodies

• Species cross-reactivity: NEURL2 demonstrates high sequence conservation across species. Antibodies show varying reactivity profiles:

  • Human-only reactive antibodies

  • Human/Mouse/Rat reactive antibodies (most common)

  • Broad reactivity antibodies (reacting with Guinea Pig, Monkey, Cow, Dog, Horse, Rabbit, etc.)

• Validation evidence: Review available validation data, including knockout controls and multiple application testing .

What are the optimal experimental conditions for NEURL2 antibody immunoblotting?

For optimal Western blotting with NEURL2 antibodies:

• Sample preparation:

  • Use standard protein extraction buffers with protease inhibitors

  • Expected molecular weight: Approximately 35 kDa

• Dilution optimization:

  • Start with manufacturer-recommended dilutions (typically 1:500-1:2000)

  • Optimize through titration experiments for specific antibody lots

• Detection system:

  • Secondary antibody selection: Anti-rabbit IgG (as most NEURL2 antibodies are rabbit-derived)

  • Both chemiluminescent and fluorescent detection systems are compatible

• Controls:

  • Positive control: Tissues/cells with known NEURL2 expression

  • Negative control: Consider peptide blocking experiments as demonstrated in validation studies

How do I validate NEURL2 antibody specificity for my experimental system?

Comprehensive validation should include:

• Genetic validation approaches:

  • Knockout/knockdown validation: Test antibody on NEURL2-deficient samples

  • Overexpression validation: Test antibody on samples with increased NEURL2 expression

• Biochemical validation:

  • Peptide competition assays: Pre-incubation with immunizing peptide should abolish specific signal

  • Multiple antibody approach: Use antibodies targeting different epitopes of NEURL2 (e.g., N-terminal vs. central regions)

• Cross-application validation:

  • Correlation between applications can be assessed, though success in one application does not guarantee performance in others

  • IF success is the best predictor of performance in WB and IP according to recent validation studies

• Species-specificity testing:

  • For evolutionary studies, verify cross-reactivity experimentally despite predicted reactivity based on sequence homology

  • Species-specific validation is critical as sequence conservation (e.g., 85% in chicken) may not translate to equal antibody affinity

How can I interpret conflicting results from different NEURL2 antibodies?

When faced with antibody discrepancies:

• Epitope consideration: Different antibodies targeting distinct regions of NEURL2 may yield varying results due to:

  • Protein conformation differences across applications

  • Post-translational modifications masking specific epitopes

  • Protein-protein interactions affecting epitope accessibility

• Methodological approach:

  • Compare antibody performance across applications systematically

  • Utilize contingency table analysis and chi-square statistics to quantify correlation between antibody performances in different applications

  • Validate key findings with orthogonal methods (e.g., mass spectrometry, RNA expression)

• Research context integration:

  • Consider biological context when interpreting results

  • Review NEURL2's role in identified genetic loci for conditions like coronary artery disease

What is known about NEURL2's biological function and expression patterns?

NEURL2 functions as an E3 ubiquitin ligase involved in protein degradation pathways. Current research indicates:

• Functional domains:

  • Contains neuralized homology domains characteristic of the neuralized family

  • Functions in ubiquitin-mediated protein degradation pathways

• Expression patterns:

  • Immunohistochemical studies show expression in human heart tissue

  • Expression has been detected in multiple cell types through Western blot analysis

• Disease associations:

  • Identified in genomic studies as potentially associated with coronary artery disease (CAD)

  • Located on chromosome 20q13.12 as part of genetic loci associated with CAD risk

How can I design experiments to study NEURL2 protein interactions?

To investigate NEURL2's interactome:

• Co-immunoprecipitation approaches:

  • Select NEURL2 antibodies validated for immunoprecipitation

  • Consider epitope location to minimize interference with protein-protein interaction sites

  • Validate interactions with reciprocal immunoprecipitation using antibodies against interacting partners

• Chromatin immunoprecipitation (ChIP) considerations:

  • For studying potential NEURL2 interactions with chromatin

  • Requires rigorous validation including controls outlined in chromatin studies

  • Consider ChIP-seq approaches for genome-wide analysis

• Proximity ligation approaches:

  • For validating interactions in situ

  • Requires two antibodies targeting different proteins

  • Consider using different host species antibodies to minimize cross-reactivity

What are common technical challenges with NEURL2 antibodies and how can they be addressed?

Researchers commonly encounter these challenges:

• Background signal issues:

  • Optimization approach: Titrate antibody concentration (start with 1:500-1:2000 for WB)

  • Blocking optimization: Test different blocking agents (BSA vs. non-fat milk)

  • Secondary antibody controls: Include no-primary antibody controls

• Inconsistent reactivity:

  • Lot-to-lot variation: Validate each new antibody lot

  • Storage issues: Follow manufacturer recommendations for storage at -20°C

  • Reconstitution: For lyophilized antibodies, reconstitute in recommended buffer (e.g., sterile distilled H₂O with 50% glycerol)

• Detection sensitivity limits:

  • Signal amplification: Consider using high-sensitivity detection systems

  • Sample enrichment: For low abundance samples, consider immunoprecipitation prior to Western blotting

  • Exposure optimization: Test multiple exposure times for optimal signal-to-noise ratio

How can I optimize NEURL2 immunohistochemistry protocols for different tissue types?

For IHC optimization:

• Antigen retrieval methods:

  • Test both heat-induced epitope retrieval (HIER) and enzymatic methods

  • Optimize pH conditions (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Adjust retrieval duration based on tissue type and fixation conditions

• Antibody incubation parameters:

  • Follow recommended dilution ranges (1:100-1:300) or (1:20-1:200)

  • Test both overnight 4°C and room temperature incubation protocols

  • Validate signal specificity with peptide competition controls

• Detection system selection:

  • For formalin-fixed paraffin-embedded tissues: Consider polymer-based detection systems

  • For frozen sections: Direct fluorescent secondary antibodies may provide optimal results

  • For dual labeling: Select compatible detection systems to minimize cross-reactivity

How is NEURL2 being studied in relation to disease mechanisms?

Current research directions include:

• Cardiovascular disease connections:

  • NEURL2 has been identified in a genomic locus (20q13.12) associated with coronary artery disease risk

  • Part of 64 novel genetic loci providing expanded view on genetic architecture of CAD

  • Antibody-based approaches can validate genetic findings at the protein level

• Neurological research applications:

  • As part of the neuralized family, NEURL2 may have roles in neurological processes

  • Antibodies enable tissue and cellular localization studies to map expression patterns

• Role in ubiquitination pathways:

  • As an E3 ubiquitin ligase , NEURL2 potentially regulates protein degradation

  • Antibody-based approaches can identify substrates and pathway components

What methodological considerations are important for multiplexed detection of NEURL2 and other proteins?

For co-localization and multiplexed studies:

• Antibody compatibility planning:

  • Host species considerations: Select antibodies from different host species to avoid cross-reactivity

  • Isotype selection: When using multiple rabbit antibodies, consider isotype-specific secondary antibodies

  • Sequential staining: For same-species antibodies, consider sequential staining with intermediate blocking steps

• Fluorescent detection optimization:

  • Spectral separation: Select fluorophores with minimal spectral overlap

  • Signal balance: Adjust antibody concentrations to achieve comparable signal intensities

  • Controls: Include single-stained controls for accurate compensation

• Advanced microscopy approaches:

  • Super-resolution techniques may reveal previously undetectable co-localization patterns

  • Live-cell imaging with fluorescently-tagged antibody fragments can monitor dynamic interactions

  • Quantitative analysis should include colocalization coefficients and statistical validation