BHLH123 Antibody

What is BHLH123 and what cellular functions does it regulate?

BHLH123 is a basic helix-loop-helix transcription factor that regulates multiple cellular processes in plants. It has been identified as a gene (also known as NAN) that encodes a transcription factor regulating cell elongation and seed germination in plants. Research has demonstrated that BHLH123 functions primarily in the nucleus, as confirmed by subcellular localization studies using fluorescence imaging of fusion proteins . The protein contains a conserved bHLH domain in its N-terminus that is critical for its DNA-binding function.

In tobacco (Nicotiana tabacum), the homologous transcription factor NtbHLH123 acts as a positive regulator and molecular switch in response to salt stress, highlighting its importance in plant stress responses . The expression of BHLH123 has been documented in various plant tissues, suggesting its broad regulatory role across different developmental stages and environmental conditions.

What are the common synonyms and identifiers for BHLH123 antibody in scientific literature?

When searching scientific literature for research involving BHLH123 antibody, researchers should be aware of multiple synonyms and identifiers used across different publications and databases. The BHLH123 antibody may be referenced using several alternative names including:

• EN63 antibody

• At3g20640 antibody (Arabidopsis thaliana gene identifier)

• F3H11.2 Transcription factor bHLH123 antibody

• Basic helix-loop-helix protein 123 antibody

• AtbHLH123 antibody (Arabidopsis thaliana specific notation)

• bHLH 123 antibody

• Transcription factor EN 63 antibody

In database searches, the UniProt accession number Q8GXT3 is the primary identifier for the BHLH123 protein. Additional database identifiers include KEGG entry ath:AT3G20640, STRING identifier 3702.AT3G20640.1, and UniGene entry At.38202. These identifiers are essential for cross-referencing research findings across different molecular biology and genomics platforms.

How does BHLH123 function as a transcription factor in gene regulation?

BHLH123 functions as a transcription factor by binding to specific DNA sequences, particularly E-box elements, in the promoter regions of target genes. The basic helix-loop-helix domain of the protein is responsible for this DNA-binding activity. Research has demonstrated that BHLH123 exhibits transactivation activity, which plays an important role in activating target genes .

In the case of NtbHLH123 (the tobacco homolog), the protein has been shown to bind specifically to E-box motifs in the promoter of the NtRbohE gene. This binding was confirmed through multiple experimental approaches including yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSA) . When NtbHLH123 binds to these regulatory elements, it can influence the transcription of genes involved in stress responses, particularly those related to salt tolerance in plants.

The regulatory function of BHLH123 involves complex protein-DNA interactions that are sequence-specific. Mutation of the E-box elements in target promoters has been shown to disrupt the binding of NtbHLH123, highlighting the specificity of this transcription factor for its cognate DNA sequences .

What are the optimal storage and handling conditions for BHLH123 antibody?

The BHLH123 antibody requires specific storage and handling conditions to maintain its functionality and specificity in experimental applications. Based on product specifications, the BHLH123 antibody is typically supplied in liquid form and should be shipped with ice packs to maintain temperature stability during transport. For long-term storage, the following conditions are recommended:

• Store at -20°C for optimal stability

• Avoid repeated freeze-thaw cycles by aliquoting the antibody into smaller volumes upon receipt

• Maintain the antibody in its buffer solution containing 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative

These storage recommendations ensure the antibody maintains its binding capacity and specificity over time. When handling the antibody for experimental use, researchers should always work with clean pipettes and sterile tubes to prevent contamination. Thawing should be done gradually on ice, and the antibody should be mixed gently by inversion or light vortexing rather than vigorous shaking to prevent protein denaturation.

How should researchers validate the specificity of BHLH123 antibody for their experimental system?

Validating antibody specificity is a critical step for ensuring reliable experimental results when working with BHLH123 antibody. Researchers should implement multiple validation approaches:

• Western blot analysis: Perform western blots using positive and negative control samples. For BHLH123, positive controls would include tissues known to express the protein (based on transcriptomic data), while negative controls might include tissues where expression is absent or knockdown/knockout cell lines.

• Immunoprecipitation followed by mass spectrometry: This approach can confirm that the antibody is pulling down the correct target protein and reveal any cross-reactivity with other proteins.

• Immunohistochemistry or immunofluorescence with blocking peptides: Compare staining patterns with and without pre-incubation of the antibody with a specific blocking peptide derived from BHLH123.

• Testing in knockout/knockdown systems: Compare antibody reactivity in wild-type versus BHLH123 knockout or knockdown samples to confirm specificity.

• Cross-validation with multiple antibodies: If available, use different antibodies targeting distinct epitopes of BHLH123 and compare results.

Researchers should also be aware that antibody specificity might vary between applications (western blot, immunoprecipitation, ChIP, etc.) and between species due to epitope conservation differences. When working with plant models other than the species for which the antibody was raised (e.g., using an Arabidopsis-specific antibody in tobacco), additional validation steps are necessary to confirm cross-reactivity.

What controls should be included in ChIP experiments using BHLH123 antibody?

Chromatin immunoprecipitation (ChIP) experiments using BHLH123 antibody require careful design of controls to ensure reliable and interpretable results. When designing ChIP experiments to study BHLH123-DNA interactions, researchers should include:

• Input DNA control: A small portion of the chromatin sample taken prior to immunoprecipitation to normalize for differences in DNA amounts and fragmentation efficiency.

• Negative control antibody: An isotype-matched non-specific antibody (such as normal IgG from the same species) to account for non-specific binding.

• Positive control antibody: An antibody against a histone mark or transcription factor known to be present in your experimental system (e.g., RNA Polymerase II or H3K4me3).

• Negative genomic region control: PCR primers targeting genomic regions not expected to bind BHLH123 (gene deserts or housekeeping gene promoters without E-box elements).

• Positive genomic region control: PCR primers targeting regions known to contain E-box elements that BHLH123 binds to, such as the NtRbohE promoter region in tobacco .

• Biological replicates: At least three independent biological replicates to account for biological variability.

When analyzing ChIP-seq data for BHLH123 binding sites, researchers should look for enrichment of E-box motifs (CANNTG) within the identified peaks, as these are the canonical binding sites for bHLH transcription factors as demonstrated in studies with NtbHLH123 .

How can BHLH123 antibody be used to study plant stress response mechanisms?

The BHLH123 antibody provides a valuable tool for investigating plant stress response mechanisms, particularly in relation to salt stress. Research has shown that the tobacco homolog NtbHLH123 functions as a positive regulator in salt stress responses by controlling Rboh-dependent mechanisms . To study these mechanisms, researchers can employ several approaches using the BHLH123 antibody:

• ChIP-seq analysis: By performing chromatin immunoprecipitation followed by high-throughput sequencing, researchers can identify the genome-wide binding sites of BHLH123 under normal and stress conditions. This approach can reveal how the transcription factor's binding profile changes in response to environmental stresses.

• Co-immunoprecipitation (Co-IP): Using the BHLH123 antibody for Co-IP experiments can identify protein interaction partners that may be involved in mediating stress responses. This is particularly valuable for understanding how BHLH123 functions within larger transcriptional complexes.

• Chromatin conformation capture (3C/4C/Hi-C): Combined with BHLH123 ChIP data, these techniques can reveal how the transcription factor influences chromatin architecture during stress responses.

• Immunoblotting across stress time courses: Western blot analysis using the BHLH123 antibody during a time course of stress treatment can reveal changes in protein abundance or post-translational modifications that may regulate BHLH123 activity.

Studies with NtbHLH123 have demonstrated that this transcription factor binds to the promoter of NtRbohE, which encodes a respiratory burst oxidase homolog involved in reactive oxygen species (ROS) production during salt stress . This finding highlights the potential of BHLH123 antibody in dissecting the molecular mechanisms of ROS signaling in plant stress responses.

What are the methodological considerations for using BHLH123 antibody in protein-DNA interaction studies?

When using BHLH123 antibody to study protein-DNA interactions, researchers must consider several methodological aspects to ensure robust and reproducible results:

• Chromatin preparation: For ChIP experiments, optimal chromatin fragmentation (200-500 bp) is critical. Sonication parameters should be optimized for each tissue or cell type being studied.

• Antibody concentration optimization: Titration experiments should be performed to determine the optimal antibody concentration that maximizes specific signal while minimizing background.

• DNA motif analysis: As BHLH123 binds to E-box elements (CANNTG), analysis of ChIP-seq data should include motif discovery to confirm enrichment of these elements in immunoprecipitated DNA.

• Validation of binding sites: After identifying potential binding sites by ChIP-seq, validation using techniques such as EMSA or reporter gene assays is recommended. For EMSA, researchers have successfully used biotin-labeled oligo-probes containing E-box motifs to demonstrate specific binding of the bHLH123 protein .

• Confirmation of binding specificity: Competition assays using unlabeled competitor probes and mutated versions of the binding site can confirm binding specificity, as demonstrated in studies of NtbHLH123 binding to the NtRbohE promoter .

• Integration with transcriptomic data: Combining ChIP-seq data with RNA-seq or microarray data can help identify direct transcriptional targets of BHLH123 and distinguish them from indirect effects.

These methodological considerations are essential for generating reliable data on BHLH123-DNA interactions and understanding the transcriptional networks regulated by this transcription factor.

How can researchers investigate BHLH123 protein interactions with other transcription factors?

Investigating protein-protein interactions (PPIs) involving BHLH123 and other transcription factors requires specialized approaches leveraging the BHLH123 antibody. Basic helix-loop-helix transcription factors often function in complexes, making these interaction studies crucial for understanding their regulatory roles. Researchers can employ the following methods:

• Co-immunoprecipitation (Co-IP): Using BHLH123 antibody to pull down the protein complex from plant cell lysates, followed by western blot or mass spectrometry analysis to identify interacting partners. This technique has been effective in identifying protein complexes in plant systems.

• Proximity ligation assay (PLA): This technique allows visualization of protein interactions in situ by using BHLH123 antibody in conjunction with antibodies against suspected interaction partners.

• Bimolecular fluorescence complementation (BiFC): While this technique uses protein tagging rather than antibodies directly, BHLH123 antibody can be used to validate expression of the fusion proteins.

• Yeast two-hybrid screening: This can identify potential interaction partners, which can then be validated in planta using Co-IP with the BHLH123 antibody.

• Sequential ChIP (Re-ChIP): This technique involves performing ChIP with BHLH123 antibody followed by a second immunoprecipitation with an antibody against a suspected partner protein to identify genomic regions where both factors co-bind.

Previous studies have shown that bHLH transcription factors often form homo- or heterodimers with other bHLH proteins or interact with transcription factors from different families to regulate gene expression . Investigating these interactions can provide insights into how BHLH123 achieves specificity in regulating different biological processes and how these interactions are modulated during stress responses.

How should researchers interpret contradictory results from BHLH123 antibody experiments across different plant species?

When researchers encounter contradictory results using BHLH123 antibody across different plant species, several factors should be considered for proper interpretation:

• Epitope conservation analysis: The sequence conservation of the epitope recognized by the BHLH123 antibody should be examined across the species being studied. Even closely related species may have subtle amino acid differences that affect antibody binding.

• Protein isoform diversity: Different plant species may express various isoforms of BHLH123 due to alternative splicing or gene duplication events. These isoforms might have different antibody reactivity profiles and functional properties.

• Post-translational modifications: Species-specific differences in post-translational modifications of BHLH123 may affect antibody recognition and protein function. Phosphorylation, ubiquitination, or other modifications could be differentially regulated across species.

• Experimental conditions: Differences in experimental conditions (extraction buffers, tissue types, developmental stages) can significantly impact antibody performance. Standardizing protocols across species comparisons is essential.

• Cross-reactivity with related proteins: The antibody may cross-react with different members of the bHLH family depending on the species, as these transcription factors share conserved domains. Western blot analysis using recombinant proteins can help assess cross-reactivity.

For example, while the Arabidopsis BHLH123 and tobacco NtbHLH123 share homology and some functional similarities, they may have evolved to regulate different target genes in their respective species . Researchers should validate their findings using complementary approaches such as RT-qPCR for transcript levels, or genetic approaches like knockdown/overexpression studies to confirm the functional role of BHLH123 in each species.

What are the common technical challenges in ChIP-seq experiments with BHLH123 antibody and how can they be addressed?

ChIP-seq experiments with BHLH123 antibody can present several technical challenges that researchers should anticipate and address:

• Low signal-to-noise ratio: BHLH123, like many transcription factors, may be expressed at relatively low levels compared to histone proteins, resulting in weak ChIP signals. This can be addressed by:

  • Increasing the amount of starting material

  • Optimizing crosslinking conditions (adjusting formaldehyde concentration and time)

  • Using more sensitive detection methods like ChIP-exo or CUT&RUN

• Antibody specificity issues: Non-specific binding can lead to false positive peaks. To mitigate this:

  • Perform stringent antibody validation as discussed earlier

  • Include appropriate controls (IgG, input DNA)

  • Use peak calling algorithms with appropriate false discovery rate controls

• DNA fragmentation inconsistencies: Uneven chromatin fragmentation can bias sequencing results. This can be improved by:

  • Standardizing sonication protocols

  • Checking fragment size distribution before proceeding to immunoprecipitation

  • Considering enzymatic fragmentation methods as alternatives

• Peak interpretation challenges: As BHLH123 binds to E-box elements (CANNTG) which are relatively common in the genome, distinguishing functional binding events from non-functional ones can be difficult. Strategies include:

  • Integrating ChIP-seq data with RNA-seq to identify binding events associated with transcriptional changes

  • Looking for co-occurrence of binding sites for known co-factors

  • Conducting motif enrichment analysis focusing on specific variants of the E-box that might be preferentially bound by BHLH123

• Experimental variability: ChIP-seq experiments can show high variability between replicates. This can be managed by:

  • Including at least three biological replicates

  • Using spike-in controls for normalization

  • Implementing robust statistical methods designed for ChIP-seq data analysis

By addressing these challenges through careful experimental design and appropriate controls, researchers can obtain more reliable and interpretable ChIP-seq data for BHLH123 binding sites.

How can researchers differentiate between direct and indirect targets of BHLH123 in transcriptional regulation studies?

Distinguishing direct from indirect targets of BHLH123 is crucial for accurately mapping its gene regulatory networks. Researchers can employ the following strategies:

• Integrated ChIP-seq and RNA-seq analysis: By combining ChIP-seq data (using BHLH123 antibody) with transcriptome profiling:

  • Direct targets are genes with BHLH123 binding sites in their regulatory regions and altered expression upon BHLH123 perturbation

  • Indirect targets show expression changes without proximal BHLH123 binding

• Time-course experiments: After inducing changes in BHLH123 activity (e.g., using inducible expression systems):

  • Direct targets typically show rapid expression changes (within hours)

  • Indirect targets respond more slowly as they require intermediate regulatory steps

• Motif analysis: Direct targets should be enriched for E-box elements in their promoters or enhancers. Specifically for BHLH123, studies of the tobacco homolog have identified specific binding to E-box motifs in target promoters such as NtRbohE .

• Transient reporter assays: Testing the ability of BHLH123 to regulate reporter gene expression driven by candidate target promoters:

  • Direct regulation should be abolished when E-box elements are mutated

  • This approach has been successfully used to validate direct binding of NtbHLH123 to the NtRbohE promoter

• Genome editing approaches: CRISPR-based editing of putative BHLH123 binding sites can determine if these sites are necessary for gene regulation:

  • If mutation of a binding site abolishes regulation, this strongly supports direct regulation

  • If regulation persists despite binding site mutation, indirect mechanisms may be involved

Using a combination of these approaches provides the strongest evidence for classifying genes as direct or indirect targets of BHLH123, helping to elucidate its primary regulatory functions versus downstream effects in signaling cascades.

How can new antibody engineering approaches improve BHLH123 antibody specificity and utility?

Recent advances in antibody engineering offer promising approaches to enhance the specificity and utility of BHLH123 antibodies for research applications:

• Single-domain antibody (VHH) technology: De novo design of single-domain antibodies using fine-tuned RFdiffusion networks represents a cutting-edge approach that could be applied to BHLH123 . These antibodies, derived from camelid heavy chain antibodies, offer several advantages:

  • Smaller size allowing better tissue penetration

  • Higher stability under various experimental conditions

  • Potential for higher specificity for particular epitopes

  • Capability to recognize epitopes inaccessible to conventional antibodies

• Recombinant antibody technology: Moving away from animal immunization to recombinant production offers:

  • More consistent batch-to-batch performance

  • Defined epitope targeting

  • Potential for sequence optimization to enhance specificity for BHLH123 over related bHLH family members

• Epitope-specific antibody development: Computational analysis of BHLH123 protein structure can identify unique epitopes that distinguish it from other bHLH family members. Antibodies raised against these unique regions would offer improved specificity.

• Nanobody-based approaches: Nanobodies against BHLH123 could be developed for applications requiring smaller detection molecules, such as super-resolution microscopy or intracellular tracking.

• Multispecificity antibodies: Engineering bispecific antibodies that recognize both BHLH123 and a common interaction partner could provide tools to specifically study functional protein complexes rather than the individual transcription factor alone.

These emerging technologies could address current limitations in BHLH123 research by providing more specific tools for distinguishing between closely related bHLH family members and enabling new experimental approaches like real-time visualization of BHLH123 activity in living cells.

What are the applications of single-cell approaches in studying BHLH123 function in heterogeneous plant tissues?

Single-cell approaches represent a frontier in plant molecular biology that can provide unprecedented insights into BHLH123 function in heterogeneous tissues:

• Single-cell RNA sequencing (scRNA-seq): This technique can reveal cell type-specific expression patterns of BHLH123 and its target genes across different cell populations within complex plant tissues. Applications include:

  • Mapping BHLH123 expression across different cell types during development

  • Identifying cell populations where BHLH123 is active during stress responses

  • Discovering cell type-specific target genes

• Single-cell ATAC-seq: By profiling chromatin accessibility, this approach can identify cell populations where BHLH123 binding sites are accessible, potentially indicating where the transcription factor is active:

  • Correlating chromatin accessibility at E-box elements with BHLH123 expression

  • Tracking changes in accessibility at BHLH123 binding sites during stress responses

• CUT&Tag at single-cell resolution: This emerging technique allows profiling of transcription factor binding at single-cell resolution and could be adapted for BHLH123:

  • Directly mapping BHLH123 binding sites in different cell types

  • Correlating binding patterns with cell-specific transcriptional responses

• Spatial transcriptomics: Techniques like Slide-seq or Visium can map BHLH123 expression and activity within the spatial context of plant tissues:

  • Identifying spatial domains of BHLH123 activity in relation to tissue architecture

  • Correlating BHLH123 expression with local microenvironments in roots or leaves

• Single-cell proteomics: Emerging single-cell proteomics methods could potentially detect BHLH123 protein levels and modifications across cell types:

  • Identifying cell populations with high BHLH123 protein abundance

  • Detecting cell type-specific post-translational modifications

These single-cell approaches can help resolve the currently limited understanding of how BHLH123 function varies across different cell types within plant tissues, potentially revealing specialized roles in specific cellular contexts that are masked in bulk tissue analyses.

How can antibody repertoire analysis techniques be applied to improve BHLH123 antibody development?

Antibody repertoire analysis techniques, such as those used in studying tumor-infiltrating B cells, can be leveraged to enhance BHLH123 antibody development:

• High-throughput B cell receptor sequencing (BCR-Seq): This technique can profile the diversity of antibodies produced in response to BHLH123 immunization:

  • Identifying highly expanded B cell clones that likely produce high-affinity antibodies against BHLH123

  • Analysis of complementarity-determining region 3 (CDR3) sequences can help identify antibodies with optimal binding properties

• Somatic hypermutation (SHM) analysis: BCR-Seq data can reveal the mutation patterns in antibody variable regions:

  • Antibodies with elevated SHM rates, as indicated by clonal polarization measures, often exhibit higher affinity and specificity

  • These highly mutated antibodies can be selected for recombinant production

• Selective B cell isolation techniques: Methods to isolate antigen-specific B cells can be applied to BHLH123 protein:

  • Flow cytometry using fluorescently labeled BHLH123 protein to isolate specific B cells

  • Single-cell sorting followed by antibody gene amplification and expression

• Computational antibody design: Using the antibody repertoire data as input:

  • Machine learning algorithms can predict optimal antibody sequences for BHLH123 binding

  • Atomically accurate de novo design approaches similar to those developed for VHH antibodies could be applied

• Epitope mapping combined with repertoire analysis: Understanding which BHLH123 epitopes elicit the strongest antibody responses:

  • Correlation between epitope recognition and antibody sequence features

  • Selection of antibodies targeting conserved epitopes for cross-species recognition

By applying these advanced antibody repertoire analysis techniques, researchers can develop next-generation BHLH123 antibodies with superior specificity, affinity, and consistency, addressing current limitations in studying this transcription factor across different plant species and experimental conditions.