NEUROD1 Antibody

What is NEUROD1 and what are its primary functions in cellular processes?

NEUROD1 is a basic helix-loop-helix (bHLH) transcription factor that functions as a transcriptional activator by binding to E-box-containing promoter consensus core sequences (5'-CANNTG-3'). It plays crucial roles in:

• Neurogenesis and terminal differentiation of neurons in the central nervous system (cerebral cortex, hippocampus, and cerebellum)

• Formation of early retinal ganglion cells and inner ear sensory neurons

• Development of endocrine islet cells in the pancreas and enteroendocrine cells in the small intestine

• Regulation of amacrine cell fate specification (in conjunction with PAX6 or SIX3)

• Dendrite morphogenesis and maintenance in the cerebellar cortex

The protein associates with the p300/CBP transcription coactivator complex to stimulate transcription of the secretin gene and the gene encoding the cyclin-dependent kinase inhibitor CDKN1A. NEUROD1 associates with chromatin at enhancer regulatory elements in genes encoding key transcriptional regulators of neurogenesis .

What are the common applications for NEUROD1 antibodies in research?

NEUROD1 antibodies are utilized in numerous research applications, with demonstrated efficacy in:

The selection of application should be based on experimental goals and validated antibody performance characteristics .

What is the observed molecular weight of NEUROD1 and how does this affect detection?

NEUROD1 has a calculated molecular weight of approximately 40 kDa, though it is often observed between 40-50 kDa on Western blots due to post-translational modifications. Key considerations include:

• The protein contains 356 amino acids with a predicted mass of 40 kDa

• Observed molecular weight can vary between 40-50 kDa depending on cell/tissue type and experimental conditions

• Post-translational modifications, particularly phosphorylation events, can affect the apparent molecular weight

• When designing experiments, researchers should include appropriate positive controls (e.g., Y79 cells, SH-SY5Y cells, or βTC-1 cells) to confirm the specificity of detection

What are the most reliable positive controls for NEUROD1 antibody validation?

For reliable validation of NEUROD1 antibodies, the following positive controls have been consistently effective:

• Cell lines: SH-SY5Y (neuroblastoma), Y79 (retinoblastoma), βTC-1/βTC-6 (mouse beta cell insulinoma)

• Tissue types: Pancreatic islets, developing cerebellum, hippocampal dentate gyrus

• Nuclear extracts: IMR32 (neuroblastoma) nuclear extracts are particularly effective

When validating a new NEUROD1 antibody, comparison with a previously validated antibody on these control samples provides the most reliable assessment of specificity and sensitivity.

How do NEUROD1 expression patterns differ during neural development and in disease models?

NEUROD1 expression follows distinct temporal and spatial patterns during development and disease states:

Neural Development:

• Early expression in developing cerebral cortex, hippocampus, and cerebellum

• Critical for terminal differentiation of neurons during late stages of neurogenesis

• Essential for granule cell formation in cerebellum and hippocampal dentate gyrus

• Contributes to development of sensory systems including retina and inner ear

Disease Models:

• Abnormal expression in neuroendocrine tumors (insulinoma, medulloblastoma, retinoblastoma, pituitary tumors)

• Expression pattern in tumors largely overlaps with IA-1 (insulinoma-associated antigen-1) expression

• Mutations in NEUROD1 cause maturity-onset diabetes of the young type VI (MODY6)

• MODY6 is characterized by autosomal dominant inheritance, onset during young adulthood, and primary insulin secretion defects

Understanding these expression patterns requires careful antibody selection and experimental design to capture the relevant developmental stages or disease phenotypes.

What is the relationship between NEUROD1 and IA-1 in gene regulation networks?

The relationship between NEUROD1 and IA-1 (insulinoma-associated antigen-1) represents a fascinating regulatory feedback loop:

• IA-1 encodes a novel zinc finger DNA-binding protein isolated from human insulinoma

• NEUROD1 regulates IA-1 gene expression through E-box elements in the IA-1 promoter

• Conversely, IA-1 functions as a transcriptional repressor that can regulate NEUROD1 gene expression

• Their expression patterns largely overlap in neuroendocrine cells, suggesting counterregulation during nervous system development

• IA-1 expression is closely associated with islet-specific transcription factors including NEUROD1

• Both proteins show restricted expression patterns in neuroendocrine tissues and tumors of neuroendocrine origin

• This regulatory network may be critical for proper development and function of neuroendocrine cells

For studying this relationship, researchers often employ chromatin immunoprecipitation, reporter gene assays, and expression analysis in various neuroendocrine cell models.

How can researchers optimize NEUROD1 antibody selection for cross-species applications?

When selecting NEUROD1 antibodies for cross-species applications, consider:

Key methodological approaches:

• Compare target epitope sequences across species before selection

• Use antibodies raised against conserved regions when possible

• Perform preliminary validation with positive control samples from the target species

• Consider using polyclonal antibodies for novel species applications as they recognize multiple epitopes

• Adjust antibody concentration and incubation conditions for optimal results in cross-species applications

What methodological considerations are important for successful NEUROD1 immunohistochemistry?

For optimal NEUROD1 immunohistochemistry results, researchers should consider:

Fixation and Antigen Retrieval:

• Formalin-fixed paraffin-embedded (FFPE) tissues require antigen retrieval

• Most effective retrieval methods include TE buffer pH 9.0 or citrate buffer pH 6.0

• Fixation time can significantly impact antibody performance; standardize protocols

Antibody Selection and Dilution:

• Polyclonal antibodies often provide stronger signal in IHC applications

• Recommended dilutions range from 1:500 to 1:2000 depending on the specific antibody

• Always include positive control tissues (pancreatic islets, developing cerebellum)

Signal Detection and Visualization:

• For fluorescent detection, NorthernLights™ 557-conjugated secondary antibodies have shown good results

• Counterstain with DAPI to visualize nuclei, as NEUROD1 is primarily nuclear

• Consider signal amplification methods for low-expression samples

Troubleshooting:

• High background: Increase blocking time and antibody dilution

• Weak signal: Optimize antigen retrieval and consider longer primary antibody incubation

• False positives: Validate with different antibody clones targeting distinct epitopes

How can researchers effectively troubleshoot negative or inconsistent NEUROD1 Western blot results?

When encountering difficulties with NEUROD1 Western blots, systematic troubleshooting should address:

Sample Preparation:

• Nuclear extraction is critical as NEUROD1 is primarily nuclear

• Include phosphatase inhibitors to preserve post-translational modifications

• Use fresh samples when possible; avoid multiple freeze-thaw cycles

Protocol Optimization:

• Adjust protein loading (25-50 μg total protein typically optimal)

• Try different transfer conditions (wet transfer may be more effective than semi-dry)

• Optimize blocking conditions (5% milk may be preferable to BSA for some antibodies)

Antibody Selection and Detection:

• Verify antibody reactivity with your species of interest

• Test multiple antibody concentrations (1:1000 to 1:5000 range)

• Consider enhanced chemiluminescence detection for improved sensitivity

Positive Controls:

• Always include a validated positive control (Y79 cells, SH-SY5Y cells)

• Recombinant NEUROD1 can serve as an additional control

• Compare results across different antibody clones when possible

Troubleshooting Decision Tree:

• No signal: Check positive control, antibody dilution, and detection system

• Multiple bands: Verify specificity with knockout/knockdown samples

• Unexpected molecular weight: Confirm with alternative antibody targeting different epitope

What are the latest advances in using NEUROD1 antibodies for tracking neuronal reprogramming?

Recent research has leveraged NEUROD1 antibodies to monitor neuronal reprogramming with several innovative approaches:

Temporal Expression Analysis:

• Sequential immunostaining to track NEUROD1 expression during transition from precursors to mature neurons

• Flow cytometry with NEUROD1 antibodies to quantify reprogramming efficiency in heterogeneous populations

• Time-course analysis correlating NEUROD1 expression with functional neuronal properties

Co-expression Studies:

• Multiplexed immunofluorescence combining NEUROD1 with other lineage markers

• Sequential staining protocols that permit visualization of up to 10 markers in the same sample

• Correlation of NEUROD1 expression with electrophysiological recordings

Chromatin Dynamics:

• ChIP-seq using NEUROD1 antibodies to map genomic binding sites during reprogramming

• Combination with ATAC-seq to correlate NEUROD1 binding with chromatin accessibility changes

• Integration with single-cell transcriptomics to link NEUROD1 activity to gene expression programs

In Vivo Applications:

• Immunohistochemistry to track reprogrammed cells in transplantation models

• Analysis of NEUROD1-mediated conversion of glial cells to neurons in injury models

• Correlation of NEUROD1 expression with functional recovery measures

These advanced applications require careful antibody validation, optimization of immunostaining protocols, and integration with complementary methodologies for comprehensive analysis .