NHLH1/NHLH2 Antibody

What are NHLH1 and NHLH2 transcription factors and what are their main functions?

NHLH1 (also known as BHLHA35, HEN1, NSCL-1) and NHLH2 (also known as BHLHA34, HEN2, NSCL-2) are closely related basic helix-loop-helix (bHLH) transcription factors that function as critical regulators of neurogenesis and neuronal differentiation. They play essential roles in the development and function of the nervous system . Both proteins share a highly homologous canonical bHLH domain in the C-terminus and a poorly conserved low-complexity domain in the N-terminus .

Between these domains, they contain a distinctive structural feature - a highly conserved stretch of 11 amino acids called the R6 domain, which encompasses six or five consecutive arginine residues immediately preceding the canonical bHLH domain . This R6 domain likely forms an extended basic domain together with the canonical basic region, giving NHLH1/2 unique DNA binding specificity.

Studies have demonstrated that NHLH1 and NHLH2 are expressed in immature neurons immediately adjacent to the ventricular zone but not in more differentiated neurons at superficial positions . They regulate the expression of genes required for neuronal differentiation and migration, including axon guidance molecules such as Robo3 . Dysregulation of these transcription factors has been implicated in neurological disorders, making them important targets for research in neurobiology and neurodevelopmental studies .

Which applications are NHLH1/NHLH2 antibodies validated for?

NHLH1/NHLH2 antibodies have been validated for several research applications, with specifications varying slightly between commercial sources. Based on the technical information from multiple suppliers, these antibodies have been validated for the following applications:

For Western blot applications, antibodies like ab72601 have demonstrated specific detection of NHLH1/NHLH2 in Jurkat cell extracts, showing a band at the expected molecular weight of 15 kDa . For immunohistochemistry, some antibodies are specifically validated for use with formalin-fixed, paraffin-embedded (FFPE) tissue sections .

It's important to note that specific applications and optimal working dilutions may vary between antibody products. For instance, Prestige Antibodies® (HPA017943) recommends dilutions of 0.04-0.4 μg/mL for Western blot applications , while others suggest ranges of 1:500-1:2000 . Researchers should consult product-specific documentation and perform optimization experiments for their particular experimental system.

How should NHLH1/NHLH2 antibodies be stored to maintain their activity?

Proper storage conditions are essential for maintaining antibody activity and specificity. Based on manufacturer recommendations for NHLH1/NHLH2 antibodies, the following storage guidelines should be followed:

• Short-term storage (up to 1 month): Store at 4°C .

• Long-term storage: Store at -20°C .

• Storage format: Most NHLH1/NHLH2 antibodies are supplied in liquid form, typically in PBS containing preservatives such as:

  • 50% glycerol

  • 0.5% BSA

  • 0.02% sodium azide

• Aliquoting: To avoid repeated freeze/thaw cycles that can degrade antibody performance, it is recommended to prepare small aliquots upon receipt of the antibody .

• Shipping conditions: These antibodies are typically shipped on wet ice or with cold packs and should be stored immediately upon receipt .

Following these storage recommendations will help ensure optimal antibody performance throughout the course of your research project, particularly for long-term studies requiring consistent antibody reactivity across multiple experiments.

What is the expected reactivity and specificity of NHLH1/NHLH2 antibodies?

NHLH1/NHLH2 antibodies demonstrate specific reactivity patterns that researchers should consider when designing experiments:

Most commercially available NHLH1/NHLH2 antibodies are raised in rabbits as polyclonal antibodies against synthetic peptides derived from human NHLH1/NHLH2 sequences . For example, the immunogen for one antibody is described as "synthesized peptide derived from the Internal region of human HEN1/2" , while another uses a synthetic peptide within the first 100 amino acids of human NHLH1 .

Regarding specificity between NHLH1 and NHLH2, it's important to note that many commercial antibodies recognize both proteins rather than distinguishing between them. This is reflected in product names such as "Anti-NHLH1/NHLH2 Antibody" or "Anti-NHLH1 + NHLH2 antibody" . This cross-reactivity is likely due to the high sequence homology between these proteins, particularly in the conserved bHLH domain.

For Western blot applications, the expected molecular weight for NHLH1/NHLH2 is approximately 15 kDa . Specificity can be verified using peptide competition, where pre-incubation of the antibody with the immunizing peptide should eliminate specific binding .

What alternative approaches can be used when antibody specificity between NHLH1 and NHLH2 is a concern?

A significant challenge in NHLH1/NHLH2 research is the difficulty in distinguishing between these two highly homologous proteins using commercially available antibodies. Multiple studies have explicitly noted that "good antibodies to detect or distinguish Nhlh1 and Nhlh2 are unavailable" . When specific detection of individual NHLH proteins is required, researchers have successfully employed these alternative approaches:

• In situ hybridization (ISH) with specific riboprobes:

  • This approach enables specific detection of NHLH1 and NHLH2 mRNAs in tissue sections

  • "Due to the lack of good antibodies for Nhlh1 and Nhlh2, we turned to ISH using Nhlh1, Nhlh2, and Robo3 riboprobes on adjacent sections"

  • ISH allows visualization of expression patterns that correlate with protein function

• RNA sequencing (RNA-seq):

  • Researchers have used RNA-seq to identify and quantify NHLH1 and NHLH2 transcripts in different cell populations

  • This approach was successfully employed to identify enrichment of NHLH1 and NHLH2 in pre-crossing pontine nucleus neurons in murine hindbrains

• Genetic approaches:

  • Generation of knockout or knockdown models for each gene individually

  • CRISPR/Cas9-mediated gene editing to create specific mutations or tagged versions

  • For example, researchers generated NHLH1/NHLH2-deficient mice by CRISPR/Cas9 to study their function in commissural neuron development

• Expression of tagged proteins:

  • Generating fusion proteins with epitope tags (e.g., HA tags) allows the use of highly specific anti-tag antibodies

  • "The HA fused or non-fused versions of Nhlh1 and Nhlh2 were confirmed to be equivalent in inducing Robo3 expression"

These methodologies, while more labor-intensive than antibody-based detection, provide more specific information about the individual roles and expression patterns of NHLH1 and NHLH2.

How can researchers validate the specificity of NHLH1/NHLH2 antibodies in their experimental systems?

Rigorous validation of NHLH1/NHLH2 antibody specificity is crucial for reliable experimental results. Here are methodological approaches for comprehensive antibody validation:

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide before application to samples

  • A specific antibody signal should be significantly reduced or eliminated when the immunizing peptide is present

  • For example, Western blot using Anti-NHLH1 + NHLH2 antibody (ab72601) with Jurkat cell extracts showed signal elimination when the immunizing peptide was included

• Genetic manipulation controls:

  • Use tissues or cells from NHLH1 or NHLH2 knockout animals as negative controls

  • Researchers have generated NHLH1/NHLH2 deficient mice that can serve as valuable negative controls

  • Test against CRISPR/Cas9-edited cell lines with known mutations in NHLH1 or NHLH2

  • Overexpression systems with tagged versions of NHLH1 or NHLH2 can serve as positive controls

• Multiple antibody validation:

  • Use antibodies from different sources that recognize different epitopes of NHLH1/NHLH2

  • Concordant results with different antibodies increase confidence in specificity

  • Compare polyclonal antibodies from different hosts or production methods

• Correlation with mRNA expression:

  • Compare antibody staining patterns with in situ hybridization results using specific riboprobes for NHLH1 and NHLH2

  • Similar expression patterns between protein and mRNA support antibody specificity

  • This approach has been successfully used in studies of neuronal development

• Western blot analysis:

  • Confirm detection of a band at the expected molecular weight (approximately 15 kDa for NHLH1/NHLH2)

  • Assess specificity across multiple cell lines or tissue types

  • Evaluate potential cross-reactivity with other bHLH family members

These validation steps are particularly important for NHLH1/NHLH2 research given the documented challenges in distinguishing between these highly homologous proteins with currently available antibodies.

What experimental approaches can be used to study NHLH1/NHLH2 function in commissural neuron development?

NHLH1 and NHLH2 play critical roles in commissural neuron development, particularly in regulating axon guidance and neuronal migration. A comprehensive experimental approach to studying their function should include:

• Temporal and spatial expression analysis:

  • Use ISH with specific riboprobes for NHLH1 and NHLH2 on adjacent sections at key developmental timepoints (E11.5-E14.5 in mice)

  • Compare expression patterns with axon guidance receptors like Robo3

  • Focus on relevant neuronal populations such as pontine nucleus neurons and spinal commissural neurons

• Gain-of-function studies:

  • Perform electroporation (EP) of NHLH1/NHLH2 expression constructs in embryonic neural tissue

  • Researchers have demonstrated that forced expression of NHLH1/2 can drive changes in axonal projection from ipsilateral to contralateral, indicating their role in determining axon laterality

  • Quantify effects on commissural axon guidance and neuronal migration

  • Assess induction of downstream targets like Robo3

• Loss-of-function analysis:

  • Examine commissural neuron development in NHLH1/NHLH2 double knockout mice

  • Studies have shown that in double mutants, pontine nucleus neurons fail to migrate towards the ventral midline and instead arrest migration in lateral positions

  • Similarly, inferior olivary nucleus neurons fail to gather tightly around the ventral midline

  • These phenotypes resemble those observed in Robo3 mutants, supporting a regulatory relationship

• Mechanistic studies:

  • Identify direct transcriptional targets using ChIP-seq or targeted ChIP-qPCR

  • Validate transcriptional regulation using reporter assays with promoter regions containing NHLH1/NHLH2 binding sites

  • Distinguish between activator and repressor functions using fusion constructs with VP16 (activator) or EnR (repressor) domains

• Structure-function analysis:

  • Generate domain deletion constructs to determine the functional importance of specific regions such as the R6 domain

  • Test the ability of mutant constructs to rescue phenotypes in knockout models

  • The R6 domain is particularly interesting as it may form an extended basic domain with the canonical basic region, potentially conferring unique DNA binding specificity

This multi-faceted approach can provide comprehensive insights into how NHLH1 and NHLH2 regulate commissural neuron development and guide axon crossing at the midline.

How do researchers identify and validate transcriptional targets of NHLH1/NHLH2?

Identifying and validating transcriptional targets of NHLH1/NHLH2 requires a systematic approach combining computational prediction, genomic analysis, and functional validation:

• Computational identification of potential binding sites:

  • Screen for NHLH1/2 binding sites in promoter/enhancer regions using position frequency matrices from databases like JASPAR

  • Researchers have successfully used this approach to identify Robo3 as a potential target: "We screened for Nhlh1/2 binding sites in the promoter/enhancer regions of genes... using the position frequency matrices of Nhlh1 and Nhlh2 from JASPAR"

  • Utilize genome browsers (e.g., UCSC) to identify conserved regions and potential regulatory elements

• Genome-wide binding site identification:

  • Perform ChIP-seq using validated NHLH1/NHLH2 antibodies or tagged versions of these proteins

  • Analyze enriched regions to identify consensus binding motifs and genomic locations

  • Example finding: "We identified a potential Nhlh1/2 binding site in the Robo3 proximal promoter (GRCm38/mm10, chr9:37,433,247-37,433,566) and four Nhlh1/2 binding sites in a distal region 26-kb 5′ to the..."

• Transcriptome analysis:

  • Compare gene expression profiles between:

    • Wild-type and NHLH1/NHLH2 knockout tissues

    • Control versus NHLH1/NHLH2 overexpressing tissues

  • RNA-seq analysis comparing pre- and post-crossing pontine nucleus neurons identified enrichment of NHLH1/2 and revealed their potential regulatory relationship with Robo3

• Functional validation:

  • Reporter assays: Clone promoter regions containing putative NHLH1/NHLH2 binding sites into reporter constructs

  • Site-directed mutagenesis of binding sites to confirm their functional significance

  • In vivo electroporation of expression constructs to assess induction of target gene expression

  • Research has shown that "forced expression of Nhlh1/2 could induce ectopic Robo3 expression" confirming the regulatory relationship

• Mechanistic characterization:

  • Determine whether NHLH1/NHLH2 function as activators or repressors for specific targets

  • Create fusion proteins with known activator domains (VP16) or repressor domains (EnR) to test functional outcomes

  • Investigate cofactors that may influence target gene regulation

  • "Evidence exists on both sides for the role of Nhlh1/2 as activators or repressors" , suggesting context-dependent regulation

This comprehensive approach has successfully identified Robo3 as a direct transcriptional target of NHLH1/NHLH2, explaining the commissural axon guidance defects observed in knockout models.

What are the implications of the unique R6 domain for NHLH1/NHLH2 antibody development and experimental design?

The R6 domain represents a distinctive structural feature of NHLH1 and NHLH2 that has significant implications for antibody development and experimental approaches:

• Structural uniqueness and functional significance:

  • The R6 domain consists of a highly conserved stretch of 11 amino acids containing six or five consecutive arginine residues

  • It is positioned immediately preceding the canonical bHLH domain

  • "The highly charged R6 domain was speculated to form a part of an extended basic domain together with the canonical basic region, rendering the DNA binding specificity unique to Nhlh1/2"

  • This domain is a feature specific to NHLH1/2 among bHLH proteins

• Challenges for antibody specificity:

  • High conservation of the R6 domain between NHLH1 and NHLH2 contributes to the difficulty in generating antibodies that can distinguish between these proteins

  • Antibodies targeting this region would likely recognize both proteins due to sequence similarity

  • The unusual amino acid composition (arginine-rich) may present technical challenges for antibody generation

• Epitope selection strategies:

  • For distinguishing between NHLH1 and NHLH2, antibodies should target less conserved regions outside the R6 domain and bHLH domain

  • For general NHLH1/NHLH2 detection, the highly conserved domains may serve as effective targets

  • Commercial antibodies often use immunogens from "Internal regions" or specific sequence regions, such as within the first 100 amino acids of human NHLH1

• Functional experimental considerations:

  • For DNA binding studies, antibodies targeting the R6 domain might interfere with protein-DNA interactions

  • For chromatin immunoprecipitation (ChIP) applications, antibodies targeting regions outside the DNA-binding domain might yield better results

  • Structure-function studies using domain deletion or mutation approaches can help elucidate the specific role of the R6 domain

• Alternative approaches when studying R6 domain function:

  • Using tagged versions of NHLH1/NHLH2 (with HA or other epitope tags) allows specific detection without relying on antibodies targeting the native proteins

  • Creating domain-specific mutations can help assess the functional importance of the R6 domain in transcriptional regulation

  • Computational modeling of protein-DNA interactions can provide insights into how the R6 domain contributes to binding specificity

Understanding the implications of the R6 domain is crucial for experimental design, particularly when studying the molecular mechanisms underlying NHLH1/NHLH2 function in neuronal development.

How should researchers design experiments to study NHLH1/NHLH2 in neurological disorders?

When investigating potential roles of NHLH1/NHLH2 in neurological disorders, researchers should implement a comprehensive experimental strategy that accounts for their developmental functions and technical limitations:

• Expression analysis in disease contexts:

  • Compare NHLH1/NHLH2 expression patterns between normal and diseased tissues using validated antibodies at appropriate dilutions

  • Implement parallel mRNA detection methods (RT-qPCR or ISH) to distinguish between NHLH1 and NHLH2 expression

  • Focus on relevant neuronal populations known to express these factors, particularly developing neurons in the ventricular and subventricular zones

• Developmental timing considerations:

  • NHLH1 and NHLH2 are primarily expressed in immature neurons during specific developmental windows

  • Select appropriate developmental timepoints (e.g., E11.5-E14.5 in mice) when studying neurodevelopmental disorders

  • For adult-onset disorders, examine whether aberrant re-expression or dysregulation occurs in adult tissues

• Functional assessment in disease models:

  • Analyze phenotypes related to neuronal migration and axon guidance in genetic models

  • NHLH1/NHLH2 double knockout mice show defects in pontine nucleus formation and inferior olivary nucleus development that resemble Robo3 mutant phenotypes

  • These defects may have implications for disorders involving abnormal brain connectivity

• Downstream target analysis:

  • Examine expression of known NHLH1/NHLH2 targets (e.g., Robo3) in disease contexts

  • Investigate whether misregulation of these targets contributes to pathology

  • Perform transcriptome analysis to identify disease-specific targets

• Genetic association studies:

  • Investigate potential associations between NHLH1/NHLH2 variants and neurological disorders

  • Focus on disorders involving commissural axon guidance defects or neuronal migration abnormalities

  • Validate functional consequences of identified variants using in vitro transcriptional assays

• Therapeutic exploration:

  • If studying NHLH1/NHLH2 for potential therapeutic applications, consider:

    • Developing more specific tools to distinguish between NHLH1 and NHLH2

    • Screening for compounds that modulate NHLH1/NHLH2 expression or activity

    • Assessing effects of interventions on downstream targets like Robo3

These methodological approaches can help elucidate potential roles of NHLH1/NHLH2 in neurological disorders and identify new therapeutic targets for conditions involving aberrant neuronal development or circuit formation.

What technical considerations are important when working with NHLH1/NHLH2 antibodies in complex neural tissues?

Working with NHLH1/NHLH2 antibodies in complex neural tissues presents several technical challenges that require careful methodological consideration:

• Tissue preparation and fixation:

  • For optimal immunohistochemistry results, standard formalin fixation (typically 4% paraformaldehyde) followed by paraffin embedding is compatible with most NHLH1/NHLH2 antibodies

  • The Sigma-Aldrich anti-NHLH1 antibody (HPA017943) is specifically validated for "immunohistochemistry (formalin-fixed, paraffin-embedded sections)"

  • Fixation time should be optimized to preserve antigenicity while maintaining tissue morphology

• Antigen retrieval and detection sensitivity:

  • For nuclear transcription factors like NHLH1/NHLH2, heat-induced epitope retrieval (HIER) is often necessary

  • Common methods include citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) heating

  • Signal amplification systems may improve detection of low-abundance transcription factors

• Specificity verification in neural tissue:

  • Include appropriate positive and negative controls in each experiment

  • Consider using tissues from NHLH1/NHLH2 knockout mice as negative controls

  • Peptide competition controls can help confirm specificity in complex neural tissues

  • Compare staining patterns with in situ hybridization results to validate expression patterns

• Cross-reactivity considerations:

  • Neural tissues express numerous bHLH transcription factors that may share structural similarities with NHLH1/NHLH2

  • Carefully validate antibody specificity in neural tissues to avoid misinterpretation

  • The highly conserved nature of the bHLH domain increases the risk of cross-reactivity

• Cellular resolution and co-localization:

  • NHLH1/NHLH2 are expressed in specific neuronal populations during development

  • Use co-labeling with neuronal markers to precisely identify expressing cell types

  • Consider confocal microscopy for accurate co-localization analysis

  • Single-cell approaches may provide higher resolution data on expression patterns

• Complementary approaches:

  • Given the limitations of antibodies in distinguishing NHLH1 from NHLH2, complement antibody-based detection with mRNA analysis

  • In situ hybridization with specific riboprobes on adjacent sections can provide valuable comparative data

  • Single-molecule fluorescence in situ hybridization (smFISH) can provide quantitative expression data at single-cell resolution

Implementing these technical considerations will improve the reliability and interpretability of NHLH1/NHLH2 antibody-based studies in complex neural tissues, particularly when investigating developmental processes or disease mechanisms.