BHLH13 Antibody

What is BHLH13 and what is its biological role?

BHLH13 (Basic Helix-Loop-Helix 13) is a transcription factor belonging to the subgroup IIId of the bHLH family in plants, particularly studied in Arabidopsis thaliana. It functions as a transcriptional repressor that negatively regulates jasmonate (JA) responses . The protein plays a critical role in mediating the balance between plant growth and defense mechanisms by interacting with JA ZIM-domain (JAZ) proteins . BHLH13 works in conjunction with other subgroup IIId factors (bHLH3, bHLH14, and bHLH17) to modulate plant responses to pathogens, insects, and various environmental stresses . Structurally, BHLH13 contains a characteristic bHLH domain in its C-terminus required for dimerization, while its N-terminus is essential for transcriptional repression function .

How is BHLH13 structurally organized and what domains are critical for antibody recognition?

BHLH13 protein contains two functionally distinct regions that should be considered when developing antibodies :

• N-terminal region (bHLH13NT): Essential for transcriptional repression activity but not involved in dimerization

• C-terminal region (bHLH13CT): Contains the bHLH domain responsible for forming homodimers and heterodimers with other subgroup IIId factors

When designing antibodies against BHLH13, researchers should consider which domain they want to target based on their experimental questions . Antibodies targeting the N-terminus may better detect the protein's functional state as a repressor, while those targeting the C-terminus might be more appropriate for studying dimerization properties or protein-protein interactions .

What is the subcellular localization of BHLH13 and how does this affect antibody selection?

BHLH13 exhibits dual localization in both the nucleus and cytoplasm, which differs from some other IIId bHLH factors like bHLH3 and bHLH17 that are primarily nucleus-localized . This dual localization pattern has significant implications for antibody-based detection experiments. When selecting or developing BHLH13 antibodies, researchers should consider whether their experimental goals require detection of the protein in specific cellular compartments . For immunofluorescence or immunohistochemistry applications, antibodies with demonstrated ability to detect both nuclear and cytoplasmic pools of the protein are preferable, as compartment-specific antibodies might provide incomplete information about BHLH13 expression and function .

What are the optimal antigen design strategies for generating BHLH13-specific antibodies?

When designing antigens for BHLH13 antibody production, researchers should consider the following evidence-based approaches:

• Domain-specific targeting: Select either the N-terminal region for function-specific antibodies or the C-terminal region for dimerization studies

• Epitope accessibility: Using bioinformatics tools to identify exposed regions of BHLH13 that are likely to be accessible in native conditions

• Unique sequence selection: Choose regions that are unique to BHLH13 and not conserved in other bHLH family members, particularly the closely related subgroup IIId members (bHLH3, bHLH14, and bHLH17)

A recommended approach includes performing sequence alignment analysis of BHLH13 against other bHLH proteins to identify unique regions with high antigenicity and surface probability scores . Peptide synthesis of these regions (typically 15-20 amino acids) conjugated to carrier proteins like KLH or BSA can serve as effective immunogens for antibody production .

What methods are most effective for producing high-specificity BHLH13 antibodies?

For producing highly specific BHLH13 antibodies, researchers can employ several approaches:

Hybridoma method:

• Immunize animals (typically mice) with purified BHLH13 protein or specific peptides

• Isolate B lymphocytes from immunized animals

• Fuse B cells with myeloma cells to form hybridomas

• Clone and screen hybridomas for specificity to BHLH13

• Expand positive clones to produce monoclonal antibodies

Single B cell method:

• Isolate B cells from immunized animals using FACS

• Extract mRNA from selected B cells

• Construct cDNA from single B cells

• Clone variable regions into expression vectors

• Screen for reactivity and specificity to BHLH13

The choice between methods depends on research requirements. Hybridoma-based strategies are well-characterized but require animal use and have relatively low efficiency at the B lymphocyte-myeloma cell fusion step . For BHLH13-specific antibodies, validation against tissue from knockout lines (e.g., bhlh13 mutants) is essential to confirm specificity and minimize cross-reactivity with other bHLH proteins .

How can researchers validate the specificity of BHLH13 antibodies?

Comprehensive validation of BHLH13 antibodies should include the following steps:

• Western blot analysis using:

  • Wild-type plant tissue expressing BHLH13

  • bhlh13 mutant tissue as a negative control

  • Tissues from plants overexpressing BHLH13 as a positive control

• Immunoprecipitation validation:

  • Using tagged versions of BHLH13 (e.g., myc-BHLH13) to confirm antibody precipitation ability

  • Performing reciprocal co-immunoprecipitation to verify protein-protein interactions

• Cross-reactivity testing:

  • Testing against other subgroup IIId bHLH proteins (bHLH3, bHLH14, bHLH17)

  • Using quadruple mutant (bhlh3 bhlh13 bhlh14 bhlh17) tissues for background assessment

• Immunolocalization consistency:

  • Verifying that subcellular localization patterns match GFP-fusion protein localization data (both nuclear and cytoplasmic for BHLH13)

A robust validation protocol should demonstrate consistent results across multiple experimental approaches and biological replicates to ensure antibody reliability for downstream applications .

How should researchers optimize Western blot protocols for BHLH13 detection?

Optimizing Western blot protocols for BHLH13 detection requires careful consideration of several parameters:

Gel selection:
Based on BHLH13's molecular weight, the following gel types are recommended:

Sample preparation:

• Include treatments that activate JA signaling (e.g., 100 μM MeJA for 40 minutes) to enhance BHLH13 expression

• Include appropriate controls:

  • Positive control: Tissue from plants overexpressing BHLH13

  • Negative control: bhlh13 mutant tissue

Blocking and antibody incubation:

• Use 5% non-fat dry milk in TBST for blocking

• Optimal antibody dilution should be determined empirically (typically 1:1000 to 1:5000)

• Incubate with primary antibody overnight at 4°C for best results

Detection considerations:
When studying BHLH13 in complex with other proteins or post-translational modifications, consider using phosphorylation-specific antibodies or performing immunoprecipitation prior to Western blot to enrich for specific interacting proteins .

What are the best approaches for using BHLH13 antibodies in chromatin immunoprecipitation (ChIP) assays?

For effective ChIP assays using BHLH13 antibodies, researchers should follow these methodological guidelines:

• Crosslinking optimization:

  • Use 1% formaldehyde for 10-15 minutes for standard crosslinking

  • For studying transient interactions, consider dual crosslinking approaches with DSG followed by formaldehyde

• Sonication parameters:

  • Aim for DNA fragments of 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis prior to immunoprecipitation

• Immunoprecipitation strategy:

  • Use at least 5 μg of BHLH13 antibody per sample

  • Include IgG control and input samples for normalization

  • Consider using plants expressing epitope-tagged BHLH13 (like myc-BHLH13) with corresponding tag antibodies for enhanced specificity

• Controls and targets:

  • Use ACTIN2 as a normalization control

  • Include primers for the 3'UTR region as a negative control

  • Target E-box or N-box containing promoters as BHLH13 binds to these cis-acting elements

• Data analysis:

  • Normalize to input DNA and IgG control

  • Calculate fold enrichment relative to control regions

  • Perform at least three biological replicates for statistical significance

Published studies have successfully employed ChIP to demonstrate BHLH13 binding to promoters of JA-responsive genes, providing a methodological framework that can be adapted for specific research questions .

How can researchers use BHLH13 antibodies to study protein-protein interactions?

BHLH13 forms both homodimers and heterodimers with other subgroup IIId bHLH factors, and interacts with JAZ proteins. To study these interactions:

• Co-immunoprecipitation (Co-IP):

  • Use plant tissues treated with MeJA to capture hormone-dependent interactions

  • Include proteasome inhibitors (e.g., MG132) to prevent degradation of JAZ proteins

  • Perform reciprocal Co-IPs to confirm interactions

  • Analyze precipitates by Western blot using antibodies against potential interacting partners

• Proximity ligation assay (PLA):

  • For in situ detection of BHLH13 interactions with other proteins

  • Requires antibodies raised in different species for BHLH13 and its interacting partners

  • Provides spatial information about where interactions occur within cells

• Bimolecular Fluorescence Complementation (BiFC):

  • Complementary to antibody-based approaches

  • Can be used to verify interactions detected by Co-IP

  • Provides information about subcellular localization of interactions

These approaches can be particularly useful for studying how BHLH13 interacts with JAZ proteins through their Jas domains and how it forms dimers via its C-terminus, providing insights into the mechanisms of JA-mediated transcriptional regulation .

What are common causes of non-specific binding with BHLH13 antibodies and how can they be addressed?

Non-specific binding is a frequent challenge when working with BHLH13 antibodies due to the conserved nature of bHLH domains across family members. Researchers can address this through:

• Cross-reactivity analysis:

  • Test antibodies against recombinant bHLH3, bHLH14, and bHLH17 proteins

  • Use tissues from single, double, triple, and quadruple mutants to identify cross-reactivity patterns

• Optimization of blocking conditions:

  • Increase blocking reagent concentration (5-10% BSA or milk)

  • Extend blocking time to 2-4 hours at room temperature

  • Add 0.1-0.5% Triton X-100 to reduce hydrophobic non-specific interactions

• Antibody pre-absorption:

  • Pre-incubate antibodies with tissue lysates from bhlh13 mutant plants

  • Remove bound antibodies by centrifugation before using the supernatant for experiments

• Dilution optimization:

  • Perform titration experiments to determine the minimum antibody concentration needed for specific detection

  • Higher dilutions often reduce background while maintaining specific signal

When non-specific binding persists, epitope mapping can help determine if the antibody recognizes conserved regions shared with other bHLH proteins, guiding the development of more specific antibodies targeted to unique regions of BHLH13 .

How can researchers overcome detection challenges when BHLH13 is expressed at low levels?

BHLH13 expression can vary depending on tissue type, developmental stage, and environmental conditions. To improve detection of low-abundance BHLH13:

• Sample enrichment techniques:

  • Use nuclear extraction protocols to concentrate nuclear-localized BHLH13

  • Employ immunoprecipitation as an enrichment step prior to Western blot

• Signal amplification methods:

  • Utilize tyramide signal amplification (TSA) for immunohistochemistry

  • Consider using high-sensitivity ECL substrates for Western blot detection

  • Implement biotin-streptavidin systems for signal enhancement

• Expression induction:

  • Treat plants with MeJA to upregulate bHLH13 expression, as it shows JA-inducible expression patterns

  • Consider using plants with constitutive promoters driving bHLH13 expression as positive controls

• Optimized extraction buffers:

  • Include protease inhibitor cocktails to prevent degradation

  • Add phosphatase inhibitors if studying phosphorylated forms

  • Use chaotropic agents or detergents optimized for nuclear proteins

The temporal expression pattern of BHLH13 should also be considered—quantitative real-time PCR can be used to identify timepoints with peak expression for optimal protein detection .

What methodological adaptations are needed for different plant species when using BHLH13 antibodies?

When adapting BHLH13 antibody protocols across different plant species, researchers should consider several factors:

• Sequence conservation analysis:

  • Perform sequence alignment of BHLH13 orthologs across target species

  • Identify conserved epitopes that antibodies might recognize

  • Consider raising species-specific antibodies for distantly related plants

• Extraction buffer optimization:

  • Adjust buffer composition based on species-specific differences in cell wall composition

  • Modify detergent concentrations for species with different membrane lipid compositions

  • Adapt protease inhibitor cocktails to species-specific proteases

• Cross-reactivity validation:

  • Test antibodies on protein extracts from multiple species

  • Perform Western blots with recombinant BHLH13 orthologs as positive controls

  • Include wild-type and mutant tissues when available

• Protocol adjustments for tissue types:

  • For woody species, include additional grinding steps and stronger extraction buffers

  • For mucilage-rich tissues, include additives to prevent interference with antibody binding

  • For tissues with high phenolic content, include PVPP or other phenolic adsorbents

A systematic validation approach when transferring BHLH13 antibody applications between species is crucial for reliable results, particularly when studying evolutionary conservation of JA signaling pathways .

How can researchers use BHLH13 antibodies to investigate its role in jasmonate signaling networks?

BHLH13 functions as a negative regulator within the jasmonate signaling pathway, and antibodies can be powerful tools for dissecting its regulatory mechanisms:

• Temporal dynamics analysis:

  • Use time-course experiments with JA treatment followed by immunoprecipitation and Western blot

  • Track changes in BHLH13 protein levels, subcellular localization, and interaction partners following JA perception

• Protein complex characterization:

  • Employ immunoprecipitation coupled with mass spectrometry (IP-MS)

  • Identify novel BHLH13-interacting proteins beyond known JAZ proteins and other bHLH factors

  • Map how these complexes change during JA responses

• Post-translational modification mapping:

  • Use modification-specific antibodies or phospho-enrichment followed by BHLH13 detection

  • Investigate how PTMs affect BHLH13 function, stability, and interactions

• ChIP-seq applications:

  • Perform genome-wide mapping of BHLH13 binding sites

  • Compare binding patterns between wild-type and JA-treated plants

  • Integrate with transcriptome data to identify direct and indirect regulatory targets

These approaches can reveal how BHLH13 coordinates with other transcription factors to fine-tune the balance between plant growth and defense responses, particularly through its antagonistic relationships with transcriptional activators like MYC2 .

What techniques combine BHLH13 antibodies with genomic approaches to understand its regulatory network?

Integrative approaches combining BHLH13 antibodies with genomic techniques provide comprehensive insights into its regulatory functions:

• ChIP-seq and CUT&RUN:

  • Use BHLH13 antibodies for genome-wide chromatin immunoprecipitation followed by sequencing

  • Identify E-box (5′-CANNTG-3′) and N-box [5′-CACG(A/C)G-3′] motifs bound by BHLH13

  • Compare binding profiles between different developmental stages or stress conditions

• ChIP-qPCR targeted validation:

  • Confirm binding to promoters of specific genes identified in ChIP-seq

  • Quantify binding affinity changes in response to JA or other treatments

  • Compare binding patterns between BHLH13 and other subgroup IIId factors

• RIP-seq (RNA immunoprecipitation sequencing):

  • Investigate potential RNA-binding capabilities of BHLH13

  • Determine if BHLH13 participates in post-transcriptional regulation

  • Identify RNA partners that might influence BHLH13 function

• Integrative multi-omics:

  • Correlate BHLH13 binding sites (ChIP-seq) with:

    • Transcriptome changes (RNA-seq) in wild-type vs. bhlh13 mutants

    • Open chromatin regions (ATAC-seq)

    • Histone modifications associated with transcriptional repression

These integrated approaches can reveal the direct targets of BHLH13 and distinguish them from secondary effects, providing a systems-level understanding of how BHLH13 coordinates with other factors to regulate JA responses .

How can BHLH13 antibodies be used to investigate crosstalk between jasmonate signaling and other hormone pathways?

Plant hormone pathways exhibit extensive crosstalk, and BHLH13 may serve as an integration node. Researchers can use BHLH13 antibodies to investigate these interactions:

• Multi-hormone treatment studies:

  • Perform immunoprecipitation after treatment with JA plus other hormones (ABA, ethylene, auxin)

  • Identify hormone-specific changes in BHLH13 interactomes

  • Track protein abundance and localization changes under different hormone combinations

• Sequential ChIP (re-ChIP):

  • Use BHLH13 antibodies in combination with antibodies against other transcription factors

  • Identify genomic regions co-bound by BHLH13 and other regulatory proteins

  • Determine if hormone crosstalk occurs at the level of chromatin binding

• Bimolecular complementation with antibody validation:

  • Use BiFC to visualize interactions between BHLH13 and components of other hormone signaling pathways

  • Validate observed interactions using co-immunoprecipitation with BHLH13 antibodies

  • Map interaction domains through deletion analysis

• Phosphorylation studies:

  • Investigate how phosphorylation of BHLH13 may be regulated by different hormone-activated kinases

  • Use phospho-specific antibodies to track BHLH13 modification status

  • Determine how phosphorylation affects BHLH13 function and interactions

Studies have shown that bHLH17/AtAIB is positively involved in ABA signaling, suggesting potential crosstalk between JA and ABA pathways that might extend to other subgroup IIId bHLH factors including BHLH13 . Antibody-based approaches can help elucidate the molecular mechanisms underlying this crosstalk.