NEUROD1 Recombinant Monoclonal Antibody
Description
In efforts to create a recombinant monoclonal antibody specific to NEUROD1, the initial step involved immunizing a rabbit with a synthesized peptide derived from human NEUROD1 protein. Following immunization, B cells were isolated from the rabbit, and RNA was extracted from these cells. The extracted RNA was reverse-transcribed into cDNA, which served as a template for extending NEUROD1 antibody genes using degenerate primers. These extended NEUROD1 antibody genes were incorporated into a plasmid vector and introduced into host cells for expression. Subsequently, the NEUROD1 recombinant monoclonal antibody was purified from the cell culture supernatant through affinity chromatography and evaluated for its utility in ELISA, IF, and FC applications, showing specific reactivity with human NEUROD1 protein.
NEUROD1 is a critical transcription factor that orchestrates the differentiation and maturation of neurons during development, contributes to the formation of neural circuits, and plays a role in the maintenance of neuronal identity. Its functions are essential for the proper functioning of the nervous system, and its dysregulation can have significant implications for neurological health and disease.
Product Specs
This recombinant monoclonal antibody, specifically targeting NEUROD1, was developed through a rigorous process. The initial step involved immunizing a rabbit with a synthesized peptide derived from human NEUROD1 protein. B cells were isolated from the immunized rabbit, and RNA was extracted. This RNA was then reverse-transcribed into cDNA, serving as a template for extending NEUROD1 antibody genes using degenerate primers. These extended genes were incorporated into a plasmid vector and introduced into host cells for expression. The resulting NEUROD1 recombinant monoclonal antibody was purified from the cell culture supernatant via affinity chromatography. Extensive testing confirmed its utility in ELISA, IF, and FC applications, demonstrating specific reactivity with human NEUROD1 protein.
NEUROD1, a crucial transcription factor, plays a pivotal role in the differentiation and maturation of neurons during development. Its involvement extends to the formation of neural circuits and the maintenance of neuronal identity. These functions are essential for the proper functioning of the nervous system, and disruptions in NEUROD1 regulation can have significant consequences for neurological health and disease.
Target Background
- Studies indicate that the first cases of MODY6 identified in Japan are attributed to missense (p.L157R, c.470T>G) or frameshift mutations (p.H206PfsTer38, c.616_617insC; p.P245RfsTer17, c.734delC; p.H206TfsTer56, c.616delC) in NEUROD1. Affected individuals and family members exhibit reduced insulin secretion capacity and neurological disorders. [CASE REPORT] PMID: 28664602
- Research suggests that the EGR1-miR-30a-5p-NEUROD1 axis may serve as a promising biomarker for diagnosing and monitoring the treatment of schizophrenic patients in acute psychotic states. EGR1 and miR-30a-5p were significantly downregulated, while NEUROD1 was upregulated in PBMNCs from patients experiencing acute psychosis. PMID: 28072411
- Mutations in the NEUROD1 gene are associated with Maturity Onset Diabetes of the Young. PMID: 28095440
- The importance of the oligomeric state of CtBP for coactivation of NeuroD1-dependent transcription was investigated. PMID: 27880001
- NeuroD1 appears insufficient to induce and maintain neuronal differentiation. Induction of neuronal differentiation by overexpression of Neurog1 initiates crucial steps for the development of glutamatergic neurons, such as spiral ganglion neurons. PMID: 27423984
- The c.133A > G (p. Ala45Thr) mutation was detected in children with sensorineural hearing loss. PMID: 26634621
- A study reports a family with autosomal dominant diabetes linked to a novel NEUROD1 mutation, one of the few meeting Maturity Onset Diabetes of the Young criteria. PMID: 26773576
- RNAi of lentiviral vector target NeuroD can reduce the migration and invasion abilities of PANC-1 cells. PMID: 24464628
- This study concludes that a novel mechanism regulates the expression of ALK in neuroblastoma and that NeuroD1 is significantly involved in neuroblastoma tumorigenesis. PMID: 25652313
- NEUROD1 is essential for maintaining retinal function, and partial loss-of-function mutations in NEUROD1 are likely a rare cause of nonsyndromic ARRP. PMID: 25477324
- Increased expression of NeuroD1 subsequently leads to the regulation of expression and function of the nicotinic acetylcholine receptor subunit cluster of alpha3, alpha5, and beta4. PMID: 24719457
- Transactivation of Ctbp was dependent on the histone H3 lysine 9 (H3K9) demethylase activity of LSD1, facilitating subsequent H3K9 acetylation by the NeuroD1-associated histone acetyltransferase, P300/CBP-associated factor. PMID: 24732800
- The variant A45T does not appear to play a major role in the development of T2 Diabetes mellitus in East Asian descent. PMID: 23203005
- Gene expression profiling revealed that permissive lines are characterized by lower expression of the early neurogenic transcription factor ASCL1 and, conversely, by higher expression of the late neurogenic transcription factor NEUROD1. PMID: 23739064
- NeuroD1 regulates survival and migration of neuroendocrine lung carcinomas via signaling molecules TrkB and NCAM. PMID: 23553831
- The overexpression of NeuroD may contribute to the tumorigenesis and development of pancreatic carcinoma, and is closely correlated with cancer cell proliferation, the p53 signal pathway, and neural invasion. PMID: 22455846
- Combined transfection of the three transcriptional factors, PDX-1, NeuroD1, and MafA, causes differentiation of bone marrow mesenchymal stem cells into insulin-producing cells. PMID: 22761608
- Photosensitive photoreceptor cells can be generated by combinations of transcription factors. The combination of CRX and RX produces immature photoreceptors; the addition of NEUROD promotes maturation. PMID: 22558175
- Most, if not all, nasal chemosensory neurons derive from NeuroD1-expressing globose basal cells of the immediate neuronal precursor variety. PMID: 21800309
- Findings establish the critical role of the neuronal differentiation factor NeuroD1 in neuroblastoma and its functional relationship with the neuronal repellent factor Slit2. PMID: 21349947
- ATF2 interacts with beta-cell-enriched transcription factors, MafA, Pdx1, and beta2, and activates insulin gene transcription. PMID: 21278380
- NeuroD alone may not be sufficient to induce regulated insulin release in insulin-producing liver cells. PMID: 21084850
- Human NeuroD1 under control of the cytokeratin 19 promoter can induce differentiation of pancreatic epithelial cells into insulin-producing cells. PMID: 20692411
- A syndrome resulting from homozygous loss of function mutations in NEUROD1 is characterized by permanent neonatal diabetes. PMID: 20573748
- There was no association between methylation and expression in breast tumor specimens, with only 14% exhibiting NEUROD1 expression. PMID: 19353266
- No significant association of NEUROD1 with retinopathy or nephropathy was found in Croatian patients with type I diabetes. PMID: 20120526
- NEUROD1 is a regulator of insulin transcription. PMID: 11755474
- NEUROD1 is expressed during trophoblast invasion. PMID: 11900979
- Beta-cell dysfunction in late-onset diabetic subjects carrying homozygous mutations in transcription factor NeuroD1. PMID: 12200761
- The genetic polymorphism in NeuroD is associated with the development of early-onset type 2 diabetes. The presence of the Thr45 allele may represent a risk factor for early-onset type 2 diabetes among Chinese. PMID: 12476420
- The Ala5Thr polymorphism of NeuroD1 is associated with Type 1 diabetes mellitus in Czech children. PMID: 12639765
- The Ala45Thr polymorphism of NeuroD1 plays a role in the risk of NIDDM in the examined Polish population. PMID: 12861411
- NeuroD1/E47 transcription factors up-regulate IA-1 gene expression through the proximal E-box element of the IA-1 promoter. PMID: 12890672
- The Ala45 variant of BETA2/NeuroD1 may be associated with IDDM in Caucasians. PMID: 12951629
- NeuroD1 is differentially expressed in pituitary adenomas, and its possible ontogenetic and/or pathogenetic implications in non-corticotroph tumors are discussed. PMID: 14759067
- No evidence of the Ala(45)Thr polymorphism of the NeuroD gene and type 1 diabetes was found. PMID: 15047635
- This review focuses on recent progress in understanding the significant role of BETA2/NeuroD1 in initiating neuronal differentiation and maintaining the nervous system. PMID: 15247487
- This polymorphism contributes to glucose intolerance in a South Indian population. PMID: 15277395
- NeuroD controls both common and distinct sets of molecules involved in cell survival and differentiation in different tissue types. [review] PMID: 15650322
- Co-expression and functional synergism of the beta-cell enriched transactivators, MafA, Pdx1, and Beta2, are critical for establishing beta-cell-specific and efficient expression of the insulin gene. PMID: 15993959
- The SREBP-1c.BETA2.E47 complex is found in a DNA looping structure, which is required for efficient recruitment of CREB-binding protein/p300. PMID: 16055439
- This study demonstrated that ISL1 and BETA2 could activate insulin gene transcription synergistically. PMID: 16321656
- A gender-specific association of the Ala45Thr variant of NEUROD1 with Type 1 diabetes was observed in Brazilian women. PMID: 16357810
- This research defines INSM1 as a transcriptional repressor of the neuroD/b2 gene. The molecular mechanism of INSM1 transcriptional repression is attributed to the recruitment of cyclin D1 and HDAC-1 and -3. PMID: 16569215
- The NeuroD1-Ala45Thr variation may have a significant role in susceptibility to or be in disequilibrium with early-onset T2DM in Chinese. The Ala45Thr may affect the onset pattern of T2DM, particularly early-onset but not late-onset T2DM in Chinese. PMID: 16773428
- The helix-loop-helix (HLH) domain of basic helix-loop-helix (bHLH) family proteins, such as NeuroD, facilitates protein transduction into various cell lines. PMID: 16870135
- Expression of NeuroD1 versus chromogranin-A is more frequent in pCA and correlates with increased indicators of malignancy in moderately to poorly differentiated pCA. PMID: 17126478
- These results suggest that NeuroD plays an important role in regulated exocytosis by inducing expressions of various components required in the process. PMID: 17217914
- A study evaluating the extent to which common variation in the six known maturity-onset diabetes of the young (MODY) genes, which cause a monogenic form of type 2 diabetes, is associated with type 2 diabetes is presented. PMID: 17327436
- Mutations in the NeuroD1/BETA2 gene contribute to the development of diabetes. PMID: 17440689
Q&A
What is NEUROD1 and what is its biological function?
NEUROD1 (Neurogenic Differentiation Factor 1) is a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors. It functions as a transcriptional activator by binding to E box-containing promoter consensus core sequences (5'-CANNTG-3'). NEUROD1 associates with the p300/CBP transcription coactivator complex to stimulate transcription of target genes including the secretin gene and cyclin-dependent kinase inhibitor CDKN1A .
NEUROD1 plays critical roles in multiple cell differentiation pathways, particularly in the development of early retinal ganglion cells, inner ear sensory neurons, cerebellar granule cells, hippocampal dentate gyrus cells, pancreatic islet cells, and enteroendocrine cells of the small intestine. Additionally, it regulates insulin gene expression, with mutations associated with type II diabetes mellitus .
What applications are NEUROD1 antibodies validated for?
NEUROD1 recombinant monoclonal antibodies have been validated for multiple research applications with specific performance characteristics:
| Application | Validated Clones | Notes |
|---|---|---|
| Western Blotting (WB) | 3H8, EPR17084 | Observed at 47 kDa (predicted 40 kDa) |
| Immunohistochemistry (IHC) | 3H8, EPR17084 | Works on both paraffin and frozen sections |
| Immunofluorescence (IF) | 3H8, EPR17084 | Shows predominantly nuclear localization |
| Flow Cytometry | 3H8 | Requires cell permeabilization |
| ELISA (E) | 3H8 | Good for quantitative analysis |
The specific applications may vary between manufacturers and clone types .
What is the difference between mouse and rabbit monoclonal antibodies for NEUROD1?
Both mouse and rabbit monoclonal antibodies are available for NEUROD1 detection, each with distinct characteristics:
| Feature | Mouse Monoclonal (e.g., 3H8) | Rabbit Monoclonal (e.g., EPR17084) |
|---|---|---|
| Host | Mouse | Rabbit |
| Isotype | IgG2a Kappa | IgG |
| Target region | aa 200-300 of human NEUROD1 | Proprietary epitope |
| Species reactivity | Human | Human, Mouse |
| Background in mouse tissues | May have higher background | Lower background in mouse tissues |
| Applications | WB, IHC-P, IF, Flow Cyt, ELISA | WB, IHC, IF |
Selection should be based on experimental requirements including target species, tissue type, and specific application .
How should NEUROD1 antibodies be stored for optimal performance?
For optimal stability and performance, NEUROD1 antibodies should be stored at -20°C or lower temperatures. To prevent degradation from repeated freeze-thaw cycles, it is recommended to aliquot the antibody into smaller volumes before freezing. Most commercially available NEUROD1 antibodies are formulated in phosphate-buffered saline (pH 7.2), sometimes with preservatives or stabilizers .
What are the optimal Western blot conditions for NEUROD1 detection?
Optimizing Western blot protocols for NEUROD1 detection requires careful consideration of several parameters:
| Parameter | Recommended Conditions |
|---|---|
| Sample preparation | Fresh tissue/cell lysates with protease inhibitors |
| Protein loading | 10-20 μg total protein per lane |
| Gel percentage | 10-12% SDS-PAGE gels |
| Transfer membrane | PVDF membrane |
| Blocking solution | 5% non-fat dry milk in TBST |
| Primary antibody dilution | 1:500-1:1000 for mouse monoclonal (3H8) |
| Primary antibody incubation | Overnight at 4°C |
| Detection system | HRP-conjugated secondary antibody with ECL |
| Expected band size | 47 kDa (observed) vs. 40 kDa (predicted) |
Most reliable positive controls include human cerebellum lysate, which shows strong NEUROD1 expression in published Western blots .
What controls should be included when working with NEUROD1 antibodies?
Proper experimental controls are essential for validating results with NEUROD1 antibodies:
Positive controls:
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Human cerebellum lysate (strongly expresses NEUROD1)
-
Human fetal heart tissue
-
Human fetal brain tissue
Negative controls:
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Mouse liver tissue (minimal NEUROD1 expression)
-
Secondary antibody-only controls (omitting primary antibody)
-
Isotype controls (irrelevant primary antibody of same isotype)
Loading/staining controls:
-
For Western blot: Anti-alpha Tubulin antibody (e.g., ab7291)
-
For immunofluorescence: DAPI nuclear counterstain
-
For IHC: Hematoxylin counterstain
Published data confirms strong NEUROD1 expression in human cerebellum but minimal expression in tissues like liver, providing reliable positive and negative tissue controls respectively .
How does epitope selection affect NEUROD1 antibody performance?
The epitope targeted by NEUROD1 antibodies significantly impacts their performance across applications:
The mouse monoclonal antibody (clone 3H8) targets amino acids 201-300 of NEUROD1, a region that includes part of the functional bHLH domain. This targeting strategy offers advantages for detecting specific functional aspects of NEUROD1 but may have limitations depending on protein conformation and interactions .
Key considerations include:
-
Epitopes within functional domains may be masked in protein complexes
-
Antibodies targeting highly conserved regions provide better cross-species reactivity
-
Post-translational modifications near the epitope may affect antibody recognition
-
Different applications (native vs. denatured protein detection) may require different epitope selections
For studying NEUROD1's role in transcriptional complexes, understanding the targeted epitope is crucial for proper experimental design and interpretation .
What immunohistochemistry protocols work best for NEUROD1 detection?
Based on published protocols, the following IHC parameters provide optimal NEUROD1 detection:
For frozen sections:
-
Fixation: 10% paraformaldehyde for 10 minutes
-
Primary antibody: Rabbit monoclonal antibody (EPR17084) at 10 μg/ml
-
Incubation: 15 minutes at room temperature
-
Detection: HRP-conjugated compact polymer system with DAB chromogen
-
Counterstain: Hematoxylin
-
Mounting: DPX mounting medium
This protocol has been successfully used for mouse brain tissue, showing strong nuclear staining in neuronal populations with minimal background .
For paraffin sections:
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Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0)
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Blocking: 5% normal serum in PBS
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Primary antibody: Mouse monoclonal antibody (3H8) at appropriate dilution
-
Detection: Standard immunoperoxidase methods
Negative control tissues (e.g., liver) should always be included to confirm specificity of staining patterns .
How can NEUROD1 antibodies be used to study neurogenesis?
NEUROD1 antibodies provide valuable tools for investigating neurogenesis in multiple contexts:
Developmental studies:
NEUROD1 is crucial for the development of neuronal populations in various regions. Antibodies can track its expression during critical developmental windows, particularly in:
-
Early retinal ganglion cells
-
Inner ear sensory neurons
-
Granule cells in the cerebellum and hippocampus
-
Pancreatic endocrine cells
Neuronal differentiation:
Experimental manipulations of NEUROD1 expression (e.g., using Neurod1-IRES-RFP constructs) demonstrate its sufficiency to promote neuroblast delamination. Co-immunostaining for NEUROD1 and neuronal markers like TuJ1 can identify stages of differentiation .
Transcriptional mechanisms:
NEUROD1 functions through binding to E-box DNA elements and association with cofactors. Chromatin immunoprecipitation using NEUROD1 antibodies can identify target genes and regulatory elements involved in neurogenesis .
Functional studies:
Research has shown that NEUROD1 can inhibit Nop-1 activity through binding to E-box elements, representing a mechanism for regulation of neural precursor differentiation. This interaction can be studied through co-immunoprecipitation and reporter assays .
What are the considerations for using NEUROD1 antibodies in flow cytometry?
Flow cytometry with NEUROD1 antibodies requires specific protocol adaptations:
Cell preparation considerations:
-
As a transcription factor, NEUROD1 is primarily nuclear, requiring cell permeabilization
-
Fixation with 2-4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 or saponin
-
Blocking with 1-5% serum matching the secondary antibody species
Antibody selection:
-
Mouse monoclonal antibody (clone 3H8) has been validated for flow cytometry
-
Starting dilution of 1:50-1:100, with titration recommended
Controls:
-
Isotype control antibodies are essential for setting proper gates
-
Include both positive control cells (neuronal precursors) and negative control populations
-
Consider co-staining with markers like TuJ1 to identify specific neuronal populations
Analysis:
-
NEUROD1 expression may be heterogeneous in developing neuronal populations
-
Consider cell cycle analysis in conjunction with NEUROD1 staining to correlate with proliferation status
The carrier-free format of some commercially available NEUROD1 antibodies makes them particularly suitable for flow cytometry applications .
