BHLH93 Antibody

What is BHLH93 and why are antibodies against it important for plant research?

BHLH93 proteins are transcription factors that play crucial roles in regulating plant developmental processes and stress responses. In apple (Malus domestica), MdbHLH93 functions as an activator of leaf senescence and directly activates the transcription of senescence-related genes such as MdSAG18 . In sugar beet, BvbHLH93 enhances salt stress tolerance by increasing antioxidant enzyme activities and reducing reactive oxygen species (ROS) generation .

Antibodies against BHLH93 are essential research tools that enable:

• Detection and quantification of BHLH93 protein levels in different tissues or under varying conditions

• Localization of BHLH93 proteins within plant cells via immunofluorescence

• Investigation of protein-protein interactions through co-immunoprecipitation

• Chromatin immunoprecipitation (ChIP) studies to identify BHLH93 binding sites on DNA

These applications provide critical insights into transcriptional regulatory networks and stress response mechanisms in plants.

How should I validate the specificity of a BHLH93 antibody before experimental use?

Methodological approach to antibody validation:

• Western blot analysis using recombinant protein: Express and purify recombinant BHLH93 protein and perform western blot to confirm antibody recognition.

• Peptide competition assay: Pre-incubate the antibody with excess synthesized peptide corresponding to the epitope, which should abolish specific binding.

• Knockout/knockdown controls: Use tissue samples from BHLH93 knockout or knockdown plants as negative controls.

• Overexpression validation: Compare signal in wild-type tissues versus tissues overexpressing BHLH93.

• Cross-reactivity assessment: Test the antibody against closely related bHLH proteins to ensure specificity within the bHLH family.

• Multiple antibody comparison: If available, compare results using different antibodies targeting different epitopes of BHLH93.

This comprehensive validation approach minimizes the risk of misinterpreting results due to non-specific antibody binding.

What are the optimal sample preparation methods for BHLH93 immunodetection in plant tissues?

For effective BHLH93 immunodetection in plant tissues:

• Tissue harvesting: Collect fresh tissue samples and immediately flash-freeze in liquid nitrogen to prevent protein degradation. For senescence studies, carefully document the developmental stage of leaves.

• Protein extraction buffer optimization:

  • Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100

  • Add protease inhibitors (e.g., PMSF, protease inhibitor cocktail)

  • Include phosphatase inhibitors if studying phosphorylation status

  • Add 10 mM DTT or β-mercaptoethanol to maintain reducing conditions

  • For nuclear proteins like BHLH93, consider specialized nuclear extraction protocols

• Homogenization: Thoroughly grind tissue in liquid nitrogen using a mortar and pestle before adding extraction buffer.

• Centrifugation steps:

  • Initial centrifugation at 15,000 × g for 15 minutes at 4°C

  • For cleaner samples, perform an additional ultracentrifugation step

• Protein quantification: Use Bradford or BCA assay to ensure equal loading in subsequent analyses.

• Sample storage: Store protein extracts at -80°C with 10% glycerol to prevent freeze-thaw damage.

These optimized methods ensure maximum preservation of BHLH93 protein integrity for subsequent immunodetection procedures.

What control samples should be included when using BHLH93 antibodies in experimental work?

A robust experimental design should include these controls:

• Positive control:

  • Recombinant BHLH93 protein

  • Tissue samples known to express high levels of BHLH93 (e.g., senescing leaves for MdbHLH93 or salt-stressed tissues for BvbHLH93 )

  • Tissues from plants overexpressing BHLH93

• Negative controls:

  • Tissues from BHLH93 knockout or knockdown plants

  • Tissues where BHLH93 expression is naturally low

  • Primary antibody omission control

  • Isotype control (using non-specific IgG of the same species)

• Treatment controls:

  • For stress studies, include both treated and untreated samples

  • For hormone response studies (e.g., ABA treatment), include appropriate vehicle controls

• Loading controls:

  • Use antibodies against housekeeping proteins (e.g., actin, tubulin)

  • For nuclear proteins, include a nuclear marker (e.g., histone H3)

These controls help validate experimental findings and provide confidence in the specificity of observed signals.

How can I optimize western blot protocols for detecting BHLH93 proteins?

Optimized western blot protocol for BHLH93 detection:

• Gel percentage selection: Use 10-12% polyacrylamide gels for optimal resolution of BHLH93 proteins.

• Protein loading: Load 20-50 μg of total protein per lane; adjust based on expression level.

• Transfer optimization:

  • Use PVDF membranes for better protein retention

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

  • Add 0.1% SDS to transfer buffer to improve large protein transfer

• Blocking optimization:

  • Block with 5% non-fat dry milk in TBST (preferred) or 3-5% BSA

  • Block for 1 hour at room temperature or overnight at 4°C

• Antibody incubation:

  • Primary antibody dilution: Start with 1:1000 and optimize as needed

  • Incubate overnight at 4°C with gentle rocking

  • Wash thoroughly (4 × 5 minutes with TBST)

  • Secondary antibody: Use 1:5000-1:10000 dilution, incubate 1 hour at room temperature

• Detection system:

  • For low abundance: Use enhanced chemiluminescence (ECL) detection

  • Consider fluorescent secondary antibodies for quantitative analysis

• Stripping and reprobing:

  • If needed, use mild stripping buffer to avoid protein loss

  • Validate complete removal of primary antibody before reprobing

This optimized protocol maximizes sensitivity while minimizing background, crucial for accurate BHLH93 detection and quantification.

How can BHLH93 antibodies be used to study protein-protein interactions in plant stress response pathways?

Methodological approaches using BHLH93 antibodies for protein interaction studies:

• Co-immunoprecipitation (Co-IP):

  • Lyse plant tissue in buffer containing mild detergents (0.5% NP-40 or Triton X-100)

  • Incubate lysate with BHLH93 antibody conjugated to protein A/G beads

  • After washing, analyze co-precipitated proteins by mass spectrometry or western blot

  • For example, this approach could identify interactions similar to the MdbHLH93-MdBT2 interaction observed in apple leaf senescence regulation

• Proximity Ligation Assay (PLA):

  • Fix and permeabilize plant tissue sections or protoplasts

  • Incubate with BHLH93 antibody and antibody against suspected interaction partner

  • Use species-specific secondary antibodies linked to complementary oligonucleotides

  • Ligase and polymerase treatment generates fluorescent signals only if proteins are in close proximity

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • Confirm antibody-identified interactions using BiFC

  • Express BHLH93 and interacting partner fused to split fluorescent protein halves

  • Reconstituted fluorescence indicates interaction

• Pull-down assays with recombinant proteins:

  • Express recombinant BHLH93 with affinity tag

  • Validate interactions identified by antibody-based methods

• Crosslinking followed by immunoprecipitation:

  • Treat plant tissues with protein crosslinkers to stabilize transient interactions

  • Immunoprecipitate using BHLH93 antibodies to capture interaction complexes

These approaches can reveal how BHLH93 interacts with other proteins to regulate stress responses, similar to how MdbHLH93 interacts with MdBT2 to regulate leaf senescence in an ABA-dependent manner .

What strategies can resolve contradictory results when using BHLH93 antibodies across different plant species?

When facing contradictory results with BHLH93 antibodies across plant species:

• Epitope conservation analysis:

  • Perform sequence alignment of BHLH93 proteins from different species

  • Determine if the antibody epitope is conserved across species

  • Design species-specific antibodies if necessary

• Antibody validation in each species:

  • Validate antibody specificity in each plant species independently

  • Use overexpression and knockout controls specific to each species

  • Perform peptide competition assays with species-specific peptides

• Multiple antibody approach:

  • Use multiple antibodies targeting different epitopes of BHLH93

  • Compare results to identify consistent patterns versus antibody-specific artifacts

• Recombinant protein standards:

  • Express recombinant BHLH93 from each species

  • Use as positive controls to determine antibody affinity differences

• Cross-linking efficiency assessment:

  • If using formaldehyde cross-linking (for ChIP), optimize conditions for each species

  • Different cell wall compositions may require adjusted protocols

• Orthogonal technique validation:

  • Complement antibody-based techniques with mRNA analysis

  • Use GFP-tagged BHLH93 expression in different species

• Data integration approach:

  • Compile results across species in standardized conditions

  • Build mathematical models to account for species-specific variations

This systematic approach can help reconcile seemingly contradictory results and highlight genuine biological differences in BHLH93 function between plant species, such as the different roles observed for MdbHLH93 in apple senescence versus BvbHLH93 in sugar beet salt tolerance .

How can I design effective ChIP-seq experiments using BHLH93 antibodies to identify genome-wide binding sites?

Comprehensive ChIP-seq experimental design for BHLH93:

• Sample preparation optimization:

  • Select tissues with high BHLH93 expression (e.g., senescing leaves for MdbHLH93 )

  • Consider inducible conditions that activate BHLH93 (e.g., salt stress for BvbHLH93 )

  • Use appropriate crosslinking (1% formaldehyde for 10 minutes)

  • Optimize sonication conditions to generate 200-500 bp DNA fragments

• ChIP protocol optimization:

  • Validate antibody specificity in ChIP conditions using known targets

  • Perform ChIP-qPCR on predicted binding sites before sequencing

  • Include input DNA and IgG controls

  • Consider using epitope-tagged BHLH93 as complementary approach

• Sequencing considerations:

  • Aim for 20-30 million reads per sample

  • Include biological replicates (minimum 3)

  • Sequence both input and ChIP samples to similar depth

• Data analysis pipeline:

  • Use appropriate peak calling algorithms (MACS2, Homer)

  • Perform motif enrichment analysis to identify BHLH93 binding motifs

  • Compare binding sites with gene expression data to identify direct targets

  • Integrate with epigenomic data (e.g., histone modifications)

• Validation strategies:

  • Confirm selected binding sites by ChIP-qPCR

  • Perform reporter assays to validate functional significance

  • Use EMSA to confirm direct binding to identified motifs

  • Validate with genetic studies (e.g., expression changes in BHLH93 mutants)

This approach would enable identification of direct targets of BHLH93, similar to how MdbHLH93 was found to directly activate MdSAG18 transcription in apple , potentially revealing the complete regulatory network of these transcription factors.

What methodologies are suitable for studying post-translational modifications of BHLH93 proteins?

Comprehensive methodologies for studying BHLH93 post-translational modifications (PTMs):

• Phosphorylation analysis:

  • Phos-tag SDS-PAGE: Incorporate Phos-tag in gels to separate phosphorylated forms

  • Phospho-specific antibodies: If available, use antibodies recognizing specific phosphorylated residues

  • Mass spectrometry: Perform LC-MS/MS analysis after immunoprecipitation

  • In vitro kinase assays: Identify kinases that modify BHLH93

• Ubiquitination analysis:

  • Immunoprecipitation under denaturing conditions: Prevents deubiquitination during extraction

  • Western blot with ubiquitin antibodies: After BHLH93 immunoprecipitation

  • Expression of tagged ubiquitin: Use HA- or His-tagged ubiquitin for pull-down experiments

  • This approach would be particularly relevant given the finding that MdBT2 induces ubiquitination and degradation of MdbHLH93 protein

• SUMOylation analysis:

  • Immunoprecipitation followed by SUMO antibody detection

  • SUMO-specific proteases (SENP) treatment: To confirm modification

  • Site-directed mutagenesis of predicted SUMOylation sites

• Glycosylation analysis:

  • Treatment with glycosidases followed by mobility shift analysis

  • Lectin blotting: To detect specific sugar moieties

  • Mass spectrometry analysis of glycosylated peptides

• Acetylation/methylation analysis:

  • Specific antibodies against acetylated or methylated lysines

  • Mass spectrometry after immunoprecipitation

  • Histone deacetylase (HDAC) or demethylase treatment

• Functional consequences assessment:

  • Site-directed mutagenesis of modified residues

  • Protein stability assays: Cycloheximide chase experiments

  • Subcellular localization studies: To determine if PTMs affect localization

  • DNA binding assays: To assess if PTMs alter DNA binding activity

These methodologies can reveal how BHLH93 activity is regulated post-translationally, similar to how MdbHLH93 is regulated by ubiquitination in response to ABA signaling .

How can BHLH93 antibodies help elucidate the regulatory mechanisms in plant stress responses?

Integrative approaches using BHLH93 antibodies to study stress regulatory networks:

• Temporal profiling of BHLH93 levels during stress exposure:

  • Monitor BHLH93 protein levels at different time points after stress application

  • Correlate changes with physiological parameters

  • For example, monitoring BvbHLH93 during progressive salt stress exposure to understand its role in enhancing antioxidant enzyme activities

• Spatial expression mapping across plant tissues:

  • Use immunohistochemistry to localize BHLH93 in different tissues

  • Compare expression patterns under normal versus stress conditions

  • Identify tissue-specific regulatory mechanisms

• Hormone-dependent regulation studies:

  • Analyze BHLH93 protein levels in response to different hormones

  • Combine with hormone biosynthesis/signaling mutants

  • Particularly relevant for studying ABA-dependent regulation of MdbHLH93

• Multi-omics integration:

  • Combine ChIP-seq data with RNA-seq to identify direct versus indirect targets

  • Integrate proteomics data to map the complete BHLH93 interactome

  • Correlate with metabolomics to understand downstream effects

• Genetic interaction mapping:

  • Compare BHLH93 protein levels and target binding in different genetic backgrounds

  • Use epistasis analysis to place BHLH93 in signaling pathways

  • For example, examining how MdbHLH93 and MdBT2 antagonistically regulate leaf senescence

• Heterologous expression studies:

  • Express BHLH93 from one species in another to assess functional conservation

  • Use antibodies to confirm expression and proper localization

  • Compare binding targets across species

This integrative approach would provide a comprehensive understanding of how BHLH93 transcription factors function in stress response networks, building on insights like the role of BvbHLH93 in enhancing salt tolerance through antioxidant enzyme regulation and MdbHLH93's function in leaf senescence .