NEUROD2 Antibody, Biotin conjugated

What is NEUROD2 and what is its biological function?

NEUROD2 is a transcriptional regulator implicated in neuronal determination during development. It mediates calcium-dependent transcription activation by binding to E box-containing promoters and serves as a critical factor in neuronal differentiation processes . NEUROD2 is primarily expressed in the postnatal cortex and mediates the activity-dependent refinement of thalamocortical axon terminals . It is mainly involved in the development of cerebellar and hippocampal granular neurons, neurons in the basolateral nucleus of amygdala, and the hypothalamic-pituitary axis . Recent research indicates that mutations in NEUROD2 can cause neurodevelopmental disorders including intellectual disability and autism spectrum disorders .

What are the molecular characteristics of NEUROD2 protein?

NEUROD2 has a calculated molecular weight of approximately 41 kDa, although in laboratory conditions it is typically observed in the 41-50 kDa range in Western blot applications . The protein is encoded by the NEUROD2 gene located on chromosome 17q12, which consists of 2 exons - exon 1 is described as a retained intron, while exon 2 contains the protein coding region . The human NEUROD2 shares high sequence homology with mouse (98.5%) and rat (97.9%) NEUROD2, making cross-species research applications possible with many antibodies .

What applications are biotin-conjugated NEUROD2 antibodies suitable for?

Biotin-conjugated NEUROD2 antibodies, such as ABIN7161287 (targeting amino acids 254-339), are primarily designed for ELISA applications . The biotin conjugation provides an advantage in detection systems that utilize avidin/streptavidin, offering amplified signal and increased sensitivity. This makes these antibodies particularly useful for detecting low-abundance NEUROD2 in complex biological samples. While ELISA is the primary application, biotin-conjugated antibodies may also be adapted for immunohistochemistry, immunocytochemistry, and flow cytometry protocols that incorporate biotin-streptavidin detection systems.

How do different NEUROD2 antibodies compare in terms of specifications and applications?

This comparison highlights the diversity of available antibodies targeting NEUROD2, with variations in host species, clonality, target regions, and applications, allowing researchers to select the most appropriate antibody for their specific experimental requirements .

What is the significance of NEUROD2 in neuronal development research?

NEUROD2 plays a crucial role in neuronal differentiation during development, making it an important target for neurodevelopmental studies . Research has shown that NEUROD2 is involved in context-dependent epigenome rewiring during neuronal differentiation . It influences chromatin accessibility in cell-type specific ways and is associated with increased global methylation in embryonic stem cells . NEUROD2 also affects three-dimensional genome organization by increasing contact frequency at binding sites and recruiting cohesin (Rad21) . These findings highlight NEUROD2's significance as a key factor in understanding the molecular mechanisms of neuronal development and neurodevelopmental disorders.

How can I optimize immunoprecipitation experiments using biotin-conjugated NEUROD2 antibodies?

For immunoprecipitation using biotin-conjugated NEUROD2 antibodies, optimization requires careful consideration of several parameters. Begin with pre-clearing your sample using protein G beads to reduce non-specific binding. When using biotin-conjugated antibodies, employ streptavidin-coated beads rather than protein A/G beads, as the biotin-streptavidin interaction is stronger and more specific.

For optimal results, use 2-5 μg of antibody per 500 μg of total protein, though this ratio may require adjustment based on NEUROD2 abundance in your specific samples. Incubate overnight at 4°C with gentle rotation to maximize antibody-antigen interaction while minimizing degradation. Include appropriate controls, especially a non-specific IgG from the same host species (rabbit for ABIN7161287), to distinguish true interactions from background .

For co-immunoprecipitation studies investigating NEUROD2's interaction partners, consider that research has demonstrated NEUROD2 interacts with chromatin modifiers and remodelers including the SWI/SNF complex subunit Brg1 (Smarca4) and Crebbp (Cbp) in a cell type-specific manner . These interactions vary between embryonic stem cells and neuronal progenitor cells, so experimental conditions should be optimized accordingly.

What are the key considerations for using NEUROD2 antibodies in chromatin immunoprecipitation (ChIP) experiments?

When designing ChIP experiments to study NEUROD2 binding to genomic regions, several factors must be considered for optimal results. While biotin-conjugated antibodies are not typically first-choice for ChIP, they can be adapted with appropriate protocol modifications.

Research has shown that NEUROD2 binds to E-box motifs in promoters, and the binding characteristics differ between cell types . When performing ChIP for NEUROD2, consider:

• Crosslinking time: NEUROD2 is a transcription factor, so standard 10-minute formaldehyde crosslinking (1% final concentration) is generally appropriate.

• Sonication conditions: Aim for chromatin fragments between 200-500 bp for optimal resolution of binding sites.

• Binding characteristics: NEUROD2 binding is enhanced at regions with multiple E-box motifs and shorter motif distances, suggesting cooperative binding . In experimental design, consider the genomic context of your regions of interest.

• Cell type specificity: NEUROD2 binding patterns show significant cell-type specificity, with differential binding observed between embryonic stem cells and neuronal progenitor cells . This must be considered when interpreting results.

• Controls: Include input controls, IgG controls, and positive controls targeting known NEUROD2 binding regions to validate your ChIP protocol.

For quantification, qPCR or sequencing approaches can be employed depending on whether you're targeting specific regions or conducting genome-wide analysis.

How does NEUROD2 expression change during neuronal differentiation and what methodological approaches can detect these changes?

NEUROD2 expression follows a complex pattern during neuronal differentiation that can be captured using multiple complementary approaches. Based on the research literature, NEUROD2 shows stage-specific expression during neuronal development .

Reporter studies indicate that NEUROD2 expression begins after NGN3 expression in endocrine progenitors, suggesting it acts downstream in the differentiation cascade . In endocrine progenitor studies, cells with bright NEUROD2 reporter signals often expressed low or no NGN3 or NKX2-2, while cells with high NGN3 or NKX2-2 showed low or no NEUROD2 reporter activity, indicating a sequential expression pattern .

To methodologically track these expression changes:

• Temporal protein analysis: Western blot time-course experiments using antibodies like 68284-1-Ig (Proteintech) can track NEUROD2 protein levels during differentiation . Recommended dilution for WB is 1:1000-1:8000.

• Immunofluorescence co-staining: Combining NEUROD2 antibodies with markers of different neuronal differentiation stages (e.g., NGN3, NKX2-2) can reveal the temporal and spatial expression patterns .

• Reporter systems: NEUROD2 reporter systems using Venus or other fluorescent proteins knocked into the NEUROD2 locus provide real-time visualization of expression dynamics .

• Single-cell RNA sequencing: This approach can reveal heterogeneity in NEUROD2 expression across differentiating cell populations and identify co-expression patterns with other developmental genes.

What are the challenges in detecting NEUROD2 in different tissue and cell types, and how can these be addressed?

Detecting NEUROD2 presents several tissue and cell type-specific challenges that require methodological adaptations. While NEUROD2 is most abundantly expressed in brain tissue (particularly cerebellum) and during neuronal development, its detection in other tissues requires optimization .

For detecting low levels of NEUROD2, biotin-conjugated antibodies offer advantages through signal amplification with streptavidin systems. The signal-to-noise ratio can be improved by optimizing blocking conditions and implementing thorough washing protocols . The Western blot detection of NEUROD2 generally shows bands between 41-50 kDa, and antibody dilutions should be optimized for each tissue type .

What is the relationship between NEUROD2 and epigenetic modifications during neuronal development?

NEUROD2 has significant interactions with epigenetic machinery that influence neuronal development. Research has revealed that NEUROD2 contributes to context-dependent epigenome rewiring during neuronal differentiation through multiple mechanisms .

At the three-dimensional genome organization level, NEUROD2 binding sites show increased contact frequency and enhanced recruitment of the cohesin component Rad21 . This effect is more pronounced in ES cells compared to NPCs. Additionally, NEUROD2 expression leads to a shift toward short-range chromatin contacts and increased chromatin insulation specifically in ES cells .

NEUROD2 also interacts directly with chromatin modifiers, including SWI/SNF complex components and the NuRD complex. Particularly notable is the cell type-specific interaction with the SWI/SNF subunit Brg1 (Smarca4) and Crebbp (Cbp), which occurs in NPCs but not in ES cells . This suggests context-dependent recruitment of epigenetic machinery by NEUROD2.

To methodologically study these interactions, researchers should consider combining NEUROD2 ChIP-seq with assays for chromatin accessibility (ATAC-seq), DNA methylation (WGBS), and three-dimensional genome organization (Hi-C or related techniques) .

What protocols should be followed for sample preparation when using biotin-conjugated NEUROD2 antibodies?

Sample preparation protocols must be optimized based on the specific application and sample type when using biotin-conjugated NEUROD2 antibodies. For protein extraction from tissues expressing NEUROD2 (such as brain, cerebellum), a gentle lysis buffer containing protease inhibitors is essential to preserve protein integrity .

For ELISA applications with the biotin-conjugated NEUROD2 antibody (ABIN7161287):

• Tissue samples: Homogenize tissues in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0) supplemented with protease inhibitors. Centrifuge at 14,000 × g for 15 minutes at 4°C to remove debris.

• Cell culture samples: Harvest cells and wash with cold PBS before lysing in RIPA buffer (10 minutes on ice). Centrifuge at 14,000 × g for 15 minutes at 4°C.

• Protein quantification: Use a Bradford or BCA assay to determine protein concentration before proceeding with antibody-based applications.

• Sample dilution: Dilute samples in appropriate assay buffer to bring the target protein concentration within the detection range of your assay.

For applications using biotin-conjugated antibodies, be aware of endogenous biotin in samples, which may cause background. Consider pre-clearing samples with streptavidin or including biotin blocking steps in your protocol .

What are the optimal storage and handling conditions for biotin-conjugated NEUROD2 antibodies?

Proper storage and handling are crucial for maintaining the functionality of biotin-conjugated NEUROD2 antibodies. Based on manufacturer recommendations for similar antibodies:

• Storage temperature: Store at -20°C for long-term preservation. The antibody is typically stable for one year from the date of receipt when stored properly .

• After reconstitution: Store at 4°C for use within one month. For longer storage, aliquot and store at -20°C for up to six months .

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly reduce antibody activity. Create small working aliquots before freezing to minimize freeze-thaw cycles .

• Buffer conditions: The antibody is typically provided in PBS with 0.02% sodium azide and may contain glycerol (often 50%) to prevent freezing damage . Maintain these conditions when diluting or aliquoting.

• Working concentration: For ELISA applications, the appropriate working dilution should be determined experimentally, but typical starting dilutions are 1:1000-1:5000 .

• Reconstitution process: Add the recommended volume of distilled water or buffer (typically 0.2 ml) to lyophilized antibody to achieve the specified concentration (e.g., 500 μg/ml) . Allow the lyophilized antibody to fully dissolve by gentle mixing, avoiding vigorous shaking or vortexing.

Biotin-conjugated antibodies are sensitive to light exposure, so minimize light exposure during handling to preserve the conjugate integrity .

How can I validate the specificity of biotin-conjugated NEUROD2 antibodies in my experimental system?

Validating antibody specificity is crucial for ensuring reliable experimental results. For biotin-conjugated NEUROD2 antibodies, implement multiple validation approaches:

• Positive and negative controls: Include samples with known NEUROD2 expression (e.g., brain tissue, particularly cerebellum) as positive controls . For negative controls, use tissues with minimal NEUROD2 expression or NEUROD2 knockout models .

• Western blot validation: Before using in ELISA, validate antibody specificity by Western blot to confirm the expected molecular weight band (41-50 kDa for NEUROD2) . While biotin-conjugated antibodies aren't optimal for Western blot, you can use an unconjugated version of the same antibody clone.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (NEUROD2 amino acids 254-339 for ABIN7161287) to block specific binding. Comparing signal with and without peptide competition helps identify non-specific binding.

• Genetic validation: If available, test the antibody in NEUROD2 knockout or knockdown systems. Research has generated NEUROD2 knockout models where the entire open reading frame was replaced with reporter genes . Absence of signal in knockout samples confirms specificity.

• Cross-reactivity assessment: If working with non-human samples, verify cross-reactivity claims. For example, ABIN7161287 is claimed to react with human NEUROD2 , while other antibodies like A07904-1 react with human, mouse, and rat .

• Comparison with alternative detection methods: Validate findings using orthogonal approaches such as mRNA detection (qPCR, RNA-seq) or reporter systems to confirm antibody results correlate with NEUROD2 expression levels.

What are the recommended protocols for using biotin-conjugated NEUROD2 antibodies in ELISA?

For optimal ELISA performance with biotin-conjugated NEUROD2 antibodies such as ABIN7161287, follow these detailed protocol recommendations:

Indirect ELISA Protocol:

• Coating: Coat 96-well plates with recombinant NEUROD2 or sample containing NEUROD2 (50-100 μl/well) diluted in carbonate-bicarbonate buffer (pH 9.6). Incubate overnight at 4°C.

• Washing: Wash 3 times with PBS-T (PBS + 0.05% Tween-20).

• Blocking: Block with 200 μl/well of blocking buffer (PBS + 1-5% BSA or non-fat milk) for 1-2 hours at room temperature.

• Primary antibody: Add an unconjugated primary anti-NEUROD2 antibody diluted in blocking buffer. Incubate for 1-2 hours at room temperature.

• Washing: Wash 3-5 times with PBS-T.

• Secondary antibody: Add biotin-conjugated NEUROD2 antibody (ABIN7161287) diluted in blocking buffer. Incubate for 1 hour at room temperature.

• Washing: Wash 3-5 times with PBS-T.

• Detection: Add streptavidin-HRP diluted in blocking buffer (typically 1:5000-1:10000). Incubate for 30 minutes at room temperature.

• Washing: Wash 3-5 times with PBS-T.

• Substrate addition: Add TMB substrate and incubate for 15-30 minutes in the dark.

• Stop reaction: Add stop solution (2N H₂SO₄).

• Reading: Measure absorbance at 450 nm with reference at 620 nm.

Sandwich ELISA Protocol (when using paired antibodies):

• Capture antibody: Coat plate with a non-biotin conjugated NEUROD2 antibody recognizing a different epitope than ABIN7161287.

• Sample addition: After blocking, add samples containing NEUROD2.

• Detection antibody: Use biotin-conjugated NEUROD2 antibody (ABIN7161287) as the detection antibody.

• Visualization: Follow steps 7-12 from the indirect ELISA protocol.

For both protocols, include a standard curve using recombinant NEUROD2 protein and appropriate positive/negative controls. Optimize antibody concentrations through titration experiments to achieve the best signal-to-noise ratio .

What approaches can be used to multiplex detection of NEUROD2 with other neuronal markers?

Multiplexed detection of NEUROD2 alongside other neuronal markers provides valuable contextual information about neuronal differentiation and function. Several methodological approaches can be employed:

• Multi-color immunofluorescence: Combine biotin-conjugated NEUROD2 antibody (visualized with streptavidin-fluorophore) with directly labeled antibodies against other neuronal markers. This approach has been used to successfully visualize NEUROD2 expression in relation to other factors like NGN3 and NKX2-2 in endocrine progenitor cells .

• Sequential immunostaining: For markers with antibodies raised in the same host species, implement sequential staining with complete blocking between rounds. This can be combined with spectral unmixing to separate overlapping fluorophore signals.

• Flow cytometry multiplexing: Combine biotin-conjugated NEUROD2 antibody (detected with streptavidin-fluorophore) with antibodies against cell surface neuronal markers for quantitative assessment of co-expression patterns. This approach can separate NEUROD2-expressing neuronal subpopulations.

• Mass cytometry (CyTOF): For high-dimensional analysis, adapt protocols to use metal-tagged antibodies against NEUROD2 and other neuronal markers, enabling simultaneous detection of 30+ markers.

• Proximity ligation assay (PLA): To detect protein-protein interactions between NEUROD2 and other factors, implement PLA using pairs of antibodies, generating fluorescent signals only when target proteins are in close proximity (<40 nm).

• Reporter systems: Utilize genetic approaches where NEUROD2 expression is reported by one fluorophore (e.g., Venus as in NEUROD2 nVenus/nVenus models) and other factors by different fluorophores . This enables live tracking of multiple factors simultaneously.

When designing multiplexed experiments, consider the temporal expression patterns of NEUROD2 and other markers. Research shows that NEUROD2 expression follows NGN3 in the differentiation cascade, with cells transitioning through states of high NGN3/low NEUROD2 to low NGN3/high NEUROD2 .

What are common challenges in NEUROD2 detection experiments and how can they be resolved?

Researchers frequently encounter several challenges when detecting NEUROD2 that require specific troubleshooting approaches:

For biotin-conjugated antibodies specifically, endogenous biotin in certain tissues (especially brain, liver, and kidney) can cause high background. Implement a biotin blocking step using avidin followed by biotin to saturate endogenous biotin before adding the biotin-conjugated antibody .

When troubleshooting ELISA applications, optimize each step independently, including antigen coating concentration, antibody dilutions, incubation times/temperatures, and washing stringency to achieve optimal signal-to-noise ratio.

How can I analyze NEUROD2 expression in relation to neuronal differentiation stages?

Analyzing NEUROD2 expression across neuronal differentiation requires integrating multiple approaches to capture the temporal and spatial dynamics accurately. Based on research findings:

• Temporal expression analysis: NEUROD2 expression is stage-specific during neuronal differentiation, following the expression of early factors like NGN3. Implement time-course sampling to capture the complete expression profile . This has revealed that cells transition from NGN3-high/NEUROD2-low to NGN3-low/NEUROD2-high states during differentiation .

• Co-expression analysis: Analyze NEUROD2 in relation to established stage-specific markers. Research shows NEUROD2 is expressed in a subset of endocrine progenitors, with some cells co-expressing NEUROD2 with NGN3 or NKX2-2, while others express only NEUROD2 or only the progenitor markers .

• Single-cell approaches: Bulk analysis can mask heterogeneity in NEUROD2 expression. Single-cell RNA-seq or reporter systems like NEUROD2 nVenus/nVenus allow identification of distinct cell states during differentiation .

• Functional context: Interpret NEUROD2 expression in relation to its function as a transcriptional regulator. Research shows NEUROD2 binding preferentially leads to gene activation, with the effect more prominent at cell-type specific peaks and correlated with binding strength .

• Epigenetic context: NEUROD2 expression should be analyzed alongside epigenetic changes, as it influences chromatin accessibility, DNA methylation, and three-dimensional genome organization in a context-dependent manner .

When interpreting results, consider the cell-type specificity of NEUROD2 function. For example, NEUROD2 interacts with the SWI/SNF subunit Brg1 (Smarca4) and Crebbp (Cbp) specifically in neuronal progenitor cells but not in embryonic stem cells .

What quantitative methods are recommended for analyzing NEUROD2 expression levels?

Accurate quantification of NEUROD2 expression requires selecting appropriate methods based on experimental objectives and sample types. Consider these validated approaches:

• Western blot quantification: For protein-level quantification, use antibodies like 68284-1-Ig (Proteintech) at 1:1000-1:8000 dilution . Normalize NEUROD2 signal to housekeeping proteins (β-actin, GAPDH) using densitometry software. This method is appropriate for comparing NEUROD2 levels between different tissues or treatment conditions.

• RT-qPCR: For mRNA quantification, design primers specific to NEUROD2 transcript (NCBI GenBank Accession: BC022481) . Normalize to stable reference genes validated for your experimental system. This approach provides high sensitivity for detecting changes in gene expression.

• Flow cytometry: For single-cell quantification, use intracellular staining protocols with NEUROD2 antibodies. This enables identification of NEUROD2-positive cell populations and determination of the percentage of cells expressing NEUROD2 at different intensities.

• Immunofluorescence intensity quantification: Measure mean fluorescence intensity of NEUROD2 staining in defined cellular compartments (primarily nuclear). This approach preserves spatial information while providing quantitative measurements.

• ELISA: For high-throughput quantification, develop a sandwich ELISA using paired NEUROD2 antibodies. This provides absolute quantification when used with a recombinant NEUROD2 standard curve.

• Reporter systems: For live monitoring, NEUROD2 reporter systems like NEUROD2 nVenus/nVenus allow quantification of fluorescence intensity as a proxy for NEUROD2 expression .

When selecting a quantification method, consider the detection threshold, dynamic range, and cellular resolution required for your experimental objectives. For studies of neuronal differentiation, methods that maintain single-cell resolution (flow cytometry, immunofluorescence, reporter systems) are particularly valuable for capturing cell-to-cell heterogeneity .

How should contradictory results between different NEUROD2 detection methods be interpreted?

Contradictory results between different NEUROD2 detection methods are not uncommon and require careful analysis to resolve. Consider these methodological interpretations when facing discrepancies:

• Transcription vs. Translation Discrepancies: Differences between RT-qPCR (mRNA) and Western blot/immunostaining (protein) results may reflect post-transcriptional regulation of NEUROD2. Research has demonstrated complex regulation of neuronal differentiation factors at multiple levels .

• Antibody Epitope Accessibility: Different antibodies target distinct regions of NEUROD2 (e.g., ABIN7161287 targets AA 254-339 ; A07904-1 targets A52-N382 ). Protein interactions, conformational changes, or post-translational modifications may mask specific epitopes while leaving others accessible.

• Detection Sensitivity Thresholds: Each method has different sensitivity thresholds. For example, ELISA with biotin-conjugated antibodies may detect lower NEUROD2 levels than standard Western blot. Apparent contradictions may reflect differences in detection limits rather than true biological variation.

• Cell Population Heterogeneity: Bulk analysis methods (Western blot, RT-qPCR) measure average expression across all cells, potentially masking subpopulation-specific patterns visible with single-cell methods. Research has shown heterogeneous NEUROD2 expression in differentiating neuronal populations .

• Temporal Dynamics: NEUROD2 expression changes during differentiation, with transient expression observed in specific developmental windows . Contradictory results may reflect different sampling timepoints relative to these expression dynamics.

When faced with contradictions, implement orthogonal validation approaches. For example, if Western blot and immunofluorescence results disagree, validate with a third method like a reporter system . Additionally, include positive and negative controls in all experiments, such as tissues with known NEUROD2 expression patterns or NEUROD2 knockout samples.

What are the latest methodological advances in studying NEUROD2 function in neuronal development?

Recent methodological advances have significantly enhanced our understanding of NEUROD2 function in neuronal development, offering new approaches for researchers:

• CRISPR/Cas9-mediated endogenous tagging: Recent studies have used CRISPR/Cas9 to replace the entire NEUROD2 open reading frame with reporter genes (e.g., H2B-Venus-3xHA) in iPSCs, enabling real-time visualization of NEUROD2 expression during differentiation without affecting cellular function .

• Integrated multi-omics approaches: Advanced studies combine NEUROD2 ChIP-seq with ATAC-seq, DNA methylation analysis, and Hi-C to comprehensively map NEUROD2's influence on the epigenome during neuronal differentiation . This integration reveals that NEUROD2 drives context-dependent epigenome rewiring during development.

• Single-cell technologies: Single-cell RNA-seq and ATAC-seq are being applied to resolve cell-to-cell heterogeneity in NEUROD2 expression and function during differentiation, revealing distinct cellular states and transition trajectories.

• Proteomics approaches: Studies implementing BioID or proximity labeling methods are identifying NEUROD2 interaction partners in a cell type-specific manner. These approaches have revealed that NEUROD2 interacts with the SWI/SNF complex subunit Brg1 (Smarca4) and Crebbp (Cbp) specifically in neuronal progenitor cells but not in embryonic stem cells .

• Functional genomics in human models: Rather than relying on mouse models alone, researchers are now studying NEUROD2 function in human iPSC-derived neurons and organoids, revealing species-specific aspects of NEUROD2 function. For example, NEUROD2 function was found to be dispensable for human pancreatic β cell development, contrasting with findings in mouse models .

• Live imaging of genome organization: Advanced microscopy techniques combined with CRISPR-based labeling systems are enabling visualization of how NEUROD2 influences chromatin architecture in living cells during differentiation, complementing genomic findings that NEUROD2 affects three-dimensional genome organization .

These methodological advances are transforming our understanding of NEUROD2's multifaceted roles in neuronal development, revealing intricate mechanisms of transcriptional regulation, epigenetic modification, and genome organization that underlie neuronal differentiation and function.